CN113552335B - Preparation method of immune electron microscope sample for positioning specific expression proteins in cryptosporidium cells in different parasitic periods - Google Patents
Preparation method of immune electron microscope sample for positioning specific expression proteins in cryptosporidium cells in different parasitic periods Download PDFInfo
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Abstract
The invention discloses a preparation method of an immune electron microscope sample for positioning specific expression proteins in cryptosporidium cells in different parasitic periods. The method comprises the steps of fixing an HCT-8 cell sample infected with cryptosporidium oocysts in glutaraldehyde-paraformaldehyde mixed fixing solution, then pre-embedding by low-melting agarose, dehydrating by ethanol gradient, penetrating by LR-White resin, embedding, polymerizing, ultrathin slicing a sample embedding block, and performing immunogold marking and heavy metal staining to obtain the cryptosporidium sample for electron microscope observation. The prepared sample has good reactive immunogenicity, good cryptosporidium form, complete structure and smooth edges of the sodium vacuole of the cryptosporidium, can see the sample of the internal structure of the cryptosporidium in the intracellular period, can be used for researching the form of the cryptosporidium in the intracellular parasitic period and the positioning of the polypide protein, is beneficial to researching the invasion mechanism, metabolic pathway and pathogenic mechanism of the cryptosporidium, and provides possibility for developing vaccines.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of an immune electron microscope sample for positioning specific expression proteins in cryptosporidium cells in different parasitic periods.
Background
Cryptosporidium (Cryptosporidium) is an important waterborne and foodborne transmission pathogen which, after entering the human body, mainly causes Cryptosporidium disease (Cryptosporidium) which is mainly diarrhea, and persistent diarrhea is caused particularly in children and immunodeficiency patients. Cryptosporidium is an intestinal parasite and is considered to be the second leading cause of diarrhea and death in children following rotavirus. And it is also a highly parasitic organism, and the complex life history is divided into a plurality of life stages, including asexual propagation and sexual propagation stages when parasitic in the host, and the propagation is discharged outside the body in the form of oocysts. Therefore, the research on the expression quantity of the same protein of the cryptosporidium in different living stages, the expression quantity of the same protein of different species and the intracellular subcellular structure positioning can provide possibility for researching the metabolic pathway and pathogenic mechanism of the cryptosporidium and developing vaccines. Despite some progress in the study of cryptosporidium, there are still many unknowns to explore. There is also increasing evidence that cryptosporidiosis occurs globally, particularly in children and people with low immunity or malnutrition, and that the results of many central studies worldwide suggest a link between cryptosporidiosis infection and subacute mortality. But now more research is needed to elucidate the mechanism of injury and the resulting health effects of cryptosporidium infection. The reason for how to explain the diarrhea in children caused by various intestinal pathogens is not clear. There are many obstacles to the development of cryptosporidiosis drugs, including the difficulty of propagation of these organisms in vitro. The new in vitro propagation method can promote transmission and can also improve the screening of new therapies and vaccines in vitro. The animal model for drug evaluation was not standardized to a high degree, and the characteristics of the target response associated with human efficacy were poor. Thus, studies of the intracellular survival phase of cryptosporidium have important implications for the mystery of the vital activities of cryptosporidium.
The observation of immune samples of cryptosporidium at the intracellular stage by a transmission electron microscope needs to overcome the following technical problems, and firstly, the structural integrity and the form close to the natural state of the cryptosporidium biological membrane and the inside are ensured. Second, the immunogenicity of Cryptosporidium and cells remains good, and the sample can have good antigen-antibody response. Furthermore, the internal structure of cryptosporidium at intracellular stages can be seen as clearly as possible in order to distinguish between different stages of survival. Finally, it is possible to make the different stages of life of cryptosporidium parasitic in the body as diverse as possible. The technique for preparing immune samples and performing transmission electron microscope observation by using a chemical method is an immune electron microscope technique. In contrast to other methods of research in cell biology, structural biology, immunoelectron microscopy imaging techniques are very demanding for samples, compared to the massive demands of radiation crystallography and nuclear magnetic resonance spectroscopy for samples.
An immunoelectron microscope is a high-precision, sensitive technique for localized analysis of antigens at the subcellular and ultrastructural levels, combining the specificity of antigen-antibody reactions with the high resolution of the electron microscope. The technology is mainly used for positioning microorganism antigens such as viruses, bacteria and the like, researching pathogenesis of immune diseases, researching ultrastructural immunocytochemistry and the like. The key to the success of the immune electron microscope technology is as follows: preserving the cell ultrastructure well; ensuring that the antigen of the detected cell or the subcellular structure thereof is in situ, and the antigenicity of the antigen is not lost; the selected immunoreagent can successfully penetrate through tissue cell structures to combine with antigen. Therefore, the preparation method of the immune electron microscope sample is improved on the basis of the common transmission electron microscope method, and a certain research means is provided for researching the invasion mechanism of the cryptosporidium. Thus, sample preparation by cryptosporidium immunoelectron microscopy is a key step in the whole process, and cryptosporidium morphology is well preserved and has good antigenicity. When the sample is subjected to section observation, the whole form of the cryptosporidium can be observed and identified, and after the antibody is incubated and heavy metal is stained, the antibody can be accurately combined with the corresponding cryptosporidium antigen and accurately positioned on the corresponding internal structure of the cryptosporidium. An important breakthrough must be made in this step of sample preparation.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary aim of the invention is to provide a preparation method of an immune electron microscope sample for positioning specific expression proteins in cryptosporidium cells in different parasitic periods. The method solves the problem that the prior art can not solve the problem of preparing the cryptosporidium immune electron microscope sample block with good immunogenicity, can keep the cryptosporidium form intact and undamaged, and is used for positioning the specific expression proteins in the cryptosporidium cells in different parasitic periods.
The aim of the invention is achieved by the following technical scheme:
a preparation method of an immunoelectron microscope sample for locating specific expression proteins in cryptosporidium cells in different parasitic periods comprises the following steps:
(1) Taking HCT-8 cells as infected cells, inoculating the oocysts of the cryptosporidium subjected to the capsule removal treatment, and culturing the cells;
(2) Adding glutaraldehyde-paraformaldehyde mixed fixing solution into the cultured cells for fixing;
(3) Adding agarose with low melting point into the fixed cell sample, uniformly mixing, and pre-embedding;
(4) Centrifuging to collect a cell sample, cooling, cutting into pieces, and rinsing;
(5) Gradient dewatering the rinsed massive cell sample with ethanol, and gradient infiltration, embedding and polymerization with LR-White resin;
(6) And (3) slicing the cell sample obtained in the step (5), performing immunogold labeling on the obtained slice, and staining the slice with heavy metal to obtain the immunoelectron microscope sample.
The HCT-8 cells described in step (1) are preferably HCT-8 cells which grow well.
The HCT-8 cells described in step (1) are preferably prepared by first pressing them into 12-well plates 4X 10 5 ～6×10 5 Individual cells/wells were cultured; more preferably, the 12-well plate is pressed 5×10 5 Individual cells/wells were cultured.
The culture medium used for the culture is preferably 1640 medium containing 10% (w/v) Fetal Bovine Serum (FBS).
The time of inoculation in the step (1) is preferably when the cell density of HCT-8 cells is 60% -80%; more preferably, the cell density is 70% to 80%.
The inoculum size of the cryptosporidium oocysts described in step (1) is preferably 1X 10 per well of a 12-well plate 6 ～2×10 6 Individual oocyst/well calculation; more preferably 1X 10 per well of a 12 well plate 6 Oocyst/well calculation.
The stripping liquid used in the stripping treatment in the step (1) is preferably sodium hypochlorite; more preferably sodium hypochlorite at a concentration of 0.4 to 0.6% (w/v); most preferred is sodium hypochlorite at a concentration of 0.5% (w/v).
The conditions of the capsule removing treatment in the step (1) are preferably ice treatment for 8-12 min; more preferably on ice for 10 minutes.
The specific procedure for cell culture described in step (1) is preferably: firstly culturing for 2-3 h, discarding uninjured cryptosporidium parvum oocysts, rinsing with Phosphate Buffered Saline (PBS), adding new culture medium, continuously culturing for 24-48 h, discarding the culture medium, and rinsing with phosphate buffered saline.
The rinsing times are all preferably 3-4 times, and each time is 1-2 minutes.
The phosphate buffer saline is preferably 1M, pH =7.3-7.5.
The medium used for the cell culture described in step (1) is preferably 1640 medium containing 2% (w/v) fetal bovine serum.
The glutaraldehyde-paraformaldehyde mixed fixed liquid in the step (2) is preferably glutaraldehyde-paraformaldehyde mixed fixed liquid with the glutaraldehyde volume fraction of 0.1-0.5%; more preferably 0.1% glutaraldehyde-4% paraformaldehyde mixed fixative.
The glutaraldehyde-paraformaldehyde mixed fixing solution in the step (2) is preferably precooled at 3-5 ℃ before being used.
The specific operation of the fixing in the step (2) is as follows: fixing for 1-2 h at 3-5 ℃, scraping the sheet, transferring to a new pipe, continuing to fix for 4-5 h, and discarding the fixing liquid.
The low-melting agarose in the step (3) is preferably a low-melting agarose solution with a mass concentration of 1.5%; more preferably, the temperature is 38-42 ℃ and the mass concentration is 1.5% of the agarose solution with low melting point.
The conditions of the centrifugation in the step (4) are preferably a temperature of 23 to 27 ℃, a rotation speed of 5000 to 7000rpm and a time of 3 to 5 minutes; more preferably, the temperature is 25 ℃, the rotation speed is 6000rpm, and the time is 3-5 minutes.
The rotor of the centrifuge is preferably subjected to a preheating treatment at 37-42 ℃ before the centrifugation described in step (4); more preferably by preheating at 37 ℃.
The cooling treatment condition in the step (4) is preferably that the temperature is 2-4 ℃ and the time is 8-12 minutes; more preferably at a temperature of 4℃for a period of 10 minutes.
The dicing in step (4) is preferably a dicing of square with a side length of about 1 mm.
The rinsing times in the step (4) are preferably 4-5 times, and each time lasts for 12-17 minutes; more preferably 4 times for 15 minutes each. The fixing liquid is washed as clean as possible.
The reagent used for the rinsing in step (4) is preferably 1M, pH =7.3 to 7.5 Phosphate Buffer (PB).
The conditions for gradient dehydration described in step (5) are preferably: 10% ethanol dehydration at 4 ℃ for 30 minutes, 20% ethanol dehydration at 4 ℃ for 30 minutes, 30% ethanol dehydration at 4 ℃ for 30 minutes, 50% ethanol dehydration at 4 ℃ for 1 hour, 70% ethanol dehydration at-20 ℃ for 1 hour, 80% ethanol dehydration at-20 ℃ for 1 hour, 90% ethanol dehydration at-20 ℃ for 1 hour, 100% ethanol dehydration at-20 ℃ for 1 hour.
The conditions of gradient permeation described in step (5) are preferably: absolute ethyl alcohol according to the volume ratio: LR-White resin = 3:1-20 ℃ permeation 2 hours, absolute ethanol: LR-White resin = 1:1-20 ℃ permeation 2 hours, absolute ethanol: LR-White resin = 1:3-20 ℃ infiltration for 2 hours, fresh LR-White resin-20 ℃ infiltration overnight, fresh LR-White resin-20 ℃ infiltration for 24 hours.
The polymerization conditions in the step (5) are preferably polymerization for 3 to 4 days at-25 ℃ to-80 ℃ under ultraviolet light with the wavelength of 315 to 370 nm; more preferably, the polymerization is carried out at-25℃for 3 to 4 days under ultraviolet light having a wavelength of 365 nm.
The thickness of the slice described in step (6) is preferably 70nm. In practice, the embedded block can be roughly trimmed to a proper sample block size by a trimming machine, then the sample is trimmed to a smooth surface and a regular quadrilateral under a microscope by using a blade and a glass knife, and then the sample is cut into ultrathin slices by using a diamond knife.
The collection of the slices in the step (6) is preferably performed by using a nickel mesh of 100-200 mesh carbon support film.
The specific operation of the immune gold labeling in the step (6) is as follows: soaking the slices with phosphate buffered saline, and standing; adding a sealing liquid for sealing; firstly adding primary anti-dilution liquid, incubating overnight at 3-5 ℃, and rinsing; then adding secondary antibody diluent, incubating for 1-2 h at 35-38 ℃, and rinsing. More preferably, the slices are infiltrated with phosphate buffered saline and allowed to stand; adding a sealing liquid for sealing; firstly adding primary anti-dilution liquid, incubating overnight at 4 ℃, and rinsing; then adding secondary antibody diluent, incubating for 1h at 37 ℃, and rinsing.
The phosphate buffered saline is preferably 1M, pH =7.3 to 7.5 phosphate buffered saline.
The standing condition is preferably 15 to 25 minutes at room temperature.
The room temperature is 20-28 ℃.
The blocking solution is preferably a Bovine Serum Albumin (BSA) solution with the concentration of 1% -2% (w/v); more preferably, the concentration is 1% (w/v) of bovine serum albumin solution.
The closing time is preferably 15-25 minutes; more preferably 20 minutes.
The primary anti-dilution solution and the secondary anti-dilution solution are preferably diluted by phosphate buffer saline containing 1% bovine serum albumin and having a concentration of 1M, pH =7.3-7.5.
The dilution ratio of the primary anti-dilution liquid is preferably 1: 100-500 parts; more preferably 1: 100-200.
The dilution ratio of the secondary anti-dilution liquid is preferably 1: 100-500 parts; more preferably 1: 100-200.
The condition of rinsing after the primary antibody incubation is preferably that the primary antibody is rinsed with phosphate buffered saline containing 1% bovine serum albumin and having a concentration of 1M, pH =7.3 to 7.5 for 4 to 5 times and 4 to 6 minutes each time.
The condition of incubation after adding the secondary antibody is preferably that phosphate buffer saline containing 1% BSA and having the concentration of 1M, pH =7.3-7.5 is firstly used for rinsing for 4-5 times, each time for 4-6 minutes, and then double distilled water is used for rinsing for 4-5 times, each time for 4-6 minutes.
The heavy metal dyeing in the step (6) is preferably uranyl acetate and lead citrate double dyeing, and the specific operation is preferably as follows: firstly, dyeing in uranyl acetate at 37 ℃ for 1-2 hours, and rinsing; and then dyeing in lead citrate for 15-20 minutes at room temperature, rinsing, airing at room temperature, taking care of sealing during dyeing, and simultaneously adding a small amount of sodium hydroxide particles around the dyed piece.
Before the uranium acetate and the lead citrate are used, the uranium acetate and the lead citrate are preferably subjected to 14000-15000 rcf centrifugal treatment for 10-15 minutes. Larger dyeing particles can be removed, and the large particles are prevented from adhering to the sample and being unfavorable for observation.
The uranium acetate is preferably a uranium acetate solution with a concentration of 2% (w/v).
The lead citrate is preferably a lead citrate solution with a concentration of 10% (w/v).
The rinsing condition after the uranium acetate is dyed is preferably that the uranium acetate is rinsed by double distilled water for 8 to 10 times, and each time is 2 to 4 minutes.
The rinsing condition after the lead citrate is dyed is preferably that the lead citrate is rinsed by double distilled water for 10 to 12 times, and each time lasts for 2 to 4 minutes.
Compared with the prior art, the invention has the following advantages and effects:
the invention provides a preparation method of an immune electron microscope immune sample for locating specific expression proteins in cryptosporidium cells in different parasitic periods. The method is to fix the cryptosporidium sample in precooled glutaraldehyde-paraformaldehyde fixing liquid to maintain the antigen activity and physiological state of cryptosporidium, and the cryptosporidium sample prepared under the condition is similar to the natural state of cryptosporidium. The cryptosporidium sample prepared by the method of the invention adopts the sample preparation step of a transmission electron microscope, can ensure that the cryptosporidium sample keeps the natural activity state when being polymerized at low temperature, adopts the form of the cryptosporidium observed by the transmission electron microscope to be more similar to the natural state, is favorable for analyzing the information of subcellular structure positioning of the protein of the cryptosporidium under physiological conditions, improves the success rate and quality of sample preparation, and does not influence the structural form of the cryptosporidium.
Drawings
FIG. 1 is a schematic flow chart of Cryptosporidium samples prepared in example 1 of the present invention.
FIG. 2 is a transmission electron microscope image of a Cryptosporidium sample prepared in example 1 of the present invention; therein, A, B, C, D is a graph of the results observed in four different fields of view (A, B, D scale 500nm, C scale 1 μm), respectively.
FIG. 3 is a transmission electron microscope image of a Cryptosporidium sample prepared in example 2 of the present invention; therein, A, B, C, D is a graph of the results observed in four different fields of view (A, D scale 500nm, B, C scale 1 μm), respectively.
FIG. 4 is a transmission electron microscope image of a Cryptosporidium sample prepared in example 3 of the present invention; therein A, B, C, D is a plot of the results observed in four different fields of view (A, B, C, D scale 500 nm), respectively.
FIG. 5 is a transmission electron microscope image of a Cryptosporidium sample prepared in comparative example 1 of the present invention; therein A, B, C, D is a plot of the results observed in four different fields of view (A, B, C, D scale 500 nm), respectively.
FIG. 6 is a transmission electron microscope image of a Cryptosporidium sample prepared in comparative example 2 of the present invention; therein, A, B, C, D is a graph of the results observed in four different fields of view (A, B scale 400nm, C, D scale 200 nm), respectively.
Detailed Description
The present invention will be further described with reference to the following specific examples and drawings, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
The main instruments involved in the following examples: carbon dioxide incubator (Thermo); biosafety cabinet (Haier); a small desk-top temperature-controllable high-speed centrifuge (eppendorf Centrifuge 5424R); refrigerator (Haier); fume hood (Haier); low temperature ultraviolet polymerization embedding box (mesoscopy instrument); the Leica block trimming machine (Leica EM TRIM); german Leica ultra-thin slicer (Leica EM UC 7); an electrothermal constant temperature drying oven (yamato DY 610C); transmission electron microscopy (Talos L120C, thermo Fisher Scientific).
The main appliances involved in the following examples: 12 orifice plate (Thermo); cell scraping (NEST); 1.5mL, 2mL centrifuge tube (Axygen); blade (eagle); nickel mesh (mesoscopy instrument); tweezers (Dumostar).
The main reagents involved in the following examples: 1640 Medium RPMI Medium 1640basic (1×) (gibco); fetal bovine serum Fetal Bovine Serum (FBS) (gibco); green streptomycin (gibco); HCT-8 cells (Shanghai cell bank of the national academy of sciences); phosphate buffered saline PBS (HyClone); 4% (w/v) sodium hypochlorite (microphone); 4% (v/v) paraformaldehyde (league); glutaraldehyde fixative (Ted Pella, usa); low melting agarose (microphone); absolute ethanol (Tianjin, century chemical Co., ltd.); LR-White (Sigma, USA); 5% (w/v) bovine serum albumin BSA (Solarbio); lead citrate (united states SPI company); uranium acetate (american SPI company); anti-Rabbit lgG (white molecular) -Gold antibody produced in goat (SIGMA, G7402-4 ML) (gold goat Anti-Rabbit); sodium hydroxide (aletin).
Example 1
1. Preparing a sample
(1) The HCT-8 cells were thawed by shaking in a water bath at 37℃rapidly, the thawed cells were transferred to 1640 medium containing 2ml of 10% (w/v) FBS, centrifuged at 800rpm for 5 minutes, the old medium was discarded, 1ml of 1640 medium containing 10% FBS was added to resuspension, and then the mixture was added to a T25 cell culture flask containing 4ml of 1640 medium containing 10% FBS, and the cells were passaged for two passages until the HCT-8 cells were grown to 80%, and the cells were obtained in a good growth state after passage of the cells for subsequent experiments. The cells were resuspended 5X 10 per well according to the method of passaging cells 5 Individual cells were seeded into 12-well plates and cultured using 1640 medium with 10% fbs.
(2) When the HCT-8 cell density is up to 70% -80%, discarding the old culture solution, rinsing 3 times with 1M, pH =7.3-7.5 PBS, adding 1640 culture medium of 2% FBS, treating on 0.5% (w/v) sodium hypochlorite ice for 10 min, adding cryptosporidium parvum oocysts rinsed 3 times with PBS into 12-well plate, and adding 1×10 to obtain the final product 6 Oocysts/well, 6 wells were inoculated. After 2 hours, uninjured cryptosporidium oocysts were discarded, rinsed 3 times with PBS, and 1 minute each time of washing, and fresh 1640 medium of 2% FBS was added again.
2. Fixing the sample
(1) After 24 hours of seeding with cryptosporidium parvum oocysts, the culture broth was discarded, rinsed 3 times with PBS for 1 minute each time, 1 mL/well of 4℃pre-chilled 0.1% glutaraldehyde fixative (80. Mu.L of 25% glutaraldehyde in 20000. Mu.L of 4% paraformaldehyde) was added, and the samples were fixed at 4℃for 4 hours.
(2) Before the sample is fixed, the cells are scraped (in order to scrape the cells into slices, the cells are fixed for 1-2 hours, otherwise, the cells are very fragile and difficult to scrape or damage after being fixed for too long), then the cells in each hole are gently sucked into a 2mL centrifuge tube by using a Pasteur pipette with a 3-4 cm suction head, and the cell quantity of one hole is filled in each tube, so that the cells are fixed for the required time continuously. Afterwards, 6 tubes of cells were recombined into a 1.5mL centrifuge tube and the fixative was discarded (by gently pouring, leaving still and then sucking off the fixative, repeated until only cell sheets remained, to reduce cell damage). Pre-embedding with 1.5% low-melting agarose cooled to below 42deg.C, rapidly mixing, centrifuging at 25deg.C at 6000rpm for 5 min, and collecting cells (centrifuging rotor preheated in oven at 37deg.C, and loading into centrifuge before centrifuging, and the same applies below).
(3) The centrifuged sample was cooled in a refrigerator at 4℃for 10 minutes, and then the cell sample with the bottom was cut into 1mm pieces 3 The sample block was picked into a 1.5mL centrifuge tube using a toothpick, and rinsed with PB (1M, pH =7.3 to 7.5) (4 times at 4 ℃ for 15 minutes each) to clean the fixative as clean as possible.
3. Embedding a sample
(1) Gradient dehydration of ethanol: 10% ethanol at 4℃for 30 min; 20% ethanol at 4℃for 30 min; 30% ethanol at 4℃for 30 min; 50% ethanol at 4 ℃ for 1h;70%, 80% and 90% ethanol at-20 ℃ for 1 hour respectively; two times each at 100% ethanol-20℃for 1 hour.
(2) Absolute ethanol: LR-white=3:1 (volume ratio, same below) permeate at-20 ℃ for 2 hours; absolute ethanol: LR-White = 1:1 permeation for 2 hours at-20 ℃; absolute ethanol LR-white=1:3 permeate for 2 hours at-20 ℃.
(3) LR-White permeated overnight at-20 ℃ (absolute no ethanol overnight).
(4) The new LR-White is changed to permeate for 24 hours at the temperature of minus 20 ℃, and then the sample is embedded by the new LR-White and polymerized for 3 to 4 days under ultraviolet (with the wavelength of 365 nm) at the temperature of minus 25 ℃.
(5) After the sample is polymerized, the whole embedded block is taken out and put into a paper bag to be stored in a constant temperature box at 25 ℃.
4. Section, immunogold labeling, staining and observation
(1) The embedded block is roughly trimmed to the appropriate sample block size using a trimming machine, and the sample is trimmed to a smooth surface and a clean quadrilateral under a microscope using a blade and a glass knife.
(2) The sample was cut into ultra-thin sections of 70nm with a diamond knife.
(3) The sections were collected using a 200 mesh carbon support membrane nickel screen.
(4) Immune gold labeling: PBS (1M, pH=7.3-7.5) is used for infiltrating the slice, and the room temperature is not less than 15 minutes; blocking the non-specific protein binding sites with 1% bsa at room temperature for 20 min; primary antibodies (MEDLE-2 polyclonal antibodies (CpMEDLE-2) were diluted in PBS containing 1% bsa of 1% 1M, pH =7.3-7.5 (disclosed in literature "Li, b.l., wu, h.z., li, n., su, j.y., jia, r.l., jiang, j.l., feng, Y.Y, xiao, l.h. (2017) Preliminary characterization of MEDLE-2,a protein potentially involved in the invasion of Cryptosporidium parvum.Frontiers in Microbiology.8,1647" in a dilution ratio of 1:200) were incubated overnight at 4 ℃; samples were rinsed 4 times for 5 minutes with PBS containing 0.1% bsa 1M, pH =7.3-7.5; the secondary antibody (goat anti-rabbit with gold) is diluted by PBS containing 1% BSA and 1M, pH =7.3-7.5 (the dilution ratio is 1:200) and incubated for 1 hour at 37 ℃; samples were rinsed 4 times for 5 minutes with PBS containing 0.1% bsa 1M, pH =7.3-7.5; reuse of ddH 2 O rinsing for 4 times, each time for 5 minutes;
(5) Heavy metal staining: uranium dyeing: 2% (w/v) uranyl acetate, at 37 ℃ for 1-2 hours; ddH 2 O rinsing for 2 min for 8 times; 10% (w/v) lead citrate, chamberWarm dyeing for 15 minutes; ddH 2 O rinsing for 2 min for 10 times; air-drying at room temperature (the two staining solutions are centrifuged for 10-15 minutes at 14000rcf before staining, larger staining particles are removed, the large particles are prevented from adhering to a sample and being unfavorable for observation, sealing is needed to be paid attention to when lead citrate is stained, and meanwhile, a small amount of sodium hydroxide particles are added around a staining piece to absorb carbon dioxide, and the following is also carried out).
(6) The results are shown in FIG. 2. Of these, the intra-cellular stage of trophoblast phase is observed in FIG. A, the intra-cellular stage of immature schizont phase is observed in FIG. B, the intra-cellular stage of mature schizont phase is observed in FIG. C (indicated by the arrow), and the intra-cellular stage of merozoite phase of type II is observed in FIG. D. From the figure, the cryptosporidium parvum in each intracellular period has good form and complete structure, the edges of the sodium vacuoles of the cryptosporidium parvum are smooth, and the internal structure of the cryptosporidium parvum in the intracellular period can be seen clearly. By gold particles we see that CpMEDLE2, a protein aggregates into a gold particle circle on the membrane of the mature one-merozoite of Cryptosporidium parvum, and that merozoites also aggregate internally, but not in significant amounts in other stages on the membrane. Overall, cpmele 2 protein was highly expressed during the merozoite phase of maturation, and the end result after antibody incubation was found to be distinct gold particle aggregation, but different numbers of phases were different and demonstrated good antigenicity of the samples.
Example 2
1. Preparing a sample
(1) The HCT-8 cells were thawed by shaking in a water bath at 37℃rapidly, the thawed cells were transferred to 1640 medium containing 2ml of 10% (w/v) FBS, centrifuged at 800rpm for 5 minutes, the old medium was discarded, 1ml of 1640 medium containing 10% FBS was added to resuspension, and then the mixture was added to a cell culture flask containing 4ml of 1640 medium containing 10% FBS for T25, and after passage of HCT-8 cells having a density of 80%, cells having a good growth state were obtained after passage of two passages of cells, and the subsequent experiments were performed. The cells were resuspended 5X 10 per well according to the method of passaging cells 5 Individual cellsInoculated into a 12-well plate, and cultured in 1640 medium containing 10% fbs.
(2) When the HCT-8 cell density is up to 60% -70%, discarding the old culture solution, rinsing 3 times with PBS, adding 1640 culture medium of 2% FBS, treating on ice with 0.5% sodium hypochlorite for 10 min, adding cryptosporidium parvum oocysts rinsed 3 times with PBS into 12-well plate, and mixing according to 1×10 6 Oocysts/well, 6 wells were inoculated. After 2 hours, uninjured cryptosporidium oocysts were discarded, rinsed 3 times with PBS and fresh 1640 medium of 2% fbs was added again.
2. Fixing the sample
(1) After 48 hours of seeding with cryptosporidium parvum oocysts, the culture broth was discarded, rinsed 3 times with PBS for 1 minute each time, 1 mL/well of 4℃pre-chilled 0.1% glutaraldehyde fixative (80. Mu.L of 25% glutaraldehyde in 20000. Mu.L of 4% paraformaldehyde) was added and the samples were fixed at 4℃for 4 hours.
(2) Before the sample is fixed, the cells are scraped (in order to scrape the cells into slices, the cells are fixed for 1-2 hours, otherwise, the cells are very fragile and difficult to scrape or damage after being fixed for too long), then the cells in each hole are gently sucked into a 2mL centrifuge tube by using a Pasteur pipette with a 3-4 cm suction head, and the cell quantity of one hole is filled in each tube, so that the cells are fixed for the required time continuously. Thereafter, 6 tubes of cells were recombined into a 1.5mL centrifuge tube and the fixative was discarded. Adding low-melting point agarose of 1.5% cooled to below 42 ℃ for pre-embedding, quickly mixing uniformly, and centrifuging at 25 ℃ at 6000rpm for 5 minutes to collect cells.
(3) Centrifuging the pre-embedded sample, cooling the centrifuged sample in a refrigerator at 4deg.C for 10 min, and cutting the cell sample with bottom into 1mm pieces 3 The sample pieces were picked into 1.5mL centrifuge tubes using toothpicks, and then rinsed with PB (4 rinses for 15 minutes each) to clean as possible the fixative.
3. Embedding a sample
(1) Gradient dehydration of ethanol: :10% ethanol at 4℃for 30 min; 20% ethanol at 4℃for 30 min; 30% ethanol at 4℃for 30 min; 50% ethanol at 4 ℃ for 1h;70%, 80% and 90% ethanol at-20 ℃ for 1 hour respectively; two times each at 100% ethanol-20℃for 1 hour.
(2) Absolute ethanol to LR-white=3:1 permeation for 2 hours at-20 ℃; absolute ethanol: LR-White = 1:1 permeation for 2 hours at-20 ℃; absolute ethanol LR-white=1:3 permeate for 2 hours at-20 ℃.
(3) LR-White was infiltrated overnight at-20 ℃.
(4) The new LR-White is changed to permeate for 24 hours at the temperature of minus 20 ℃, and then the sample is embedded by the new LR-White and polymerized for 3 to 4 days under ultraviolet (with the wavelength of 365 nm) at the temperature of minus 25 ℃.
(5) After the sample is polymerized, the whole embedded block is taken down and put into a paper bag to be stored in a constant temperature box at 25 ℃.
4. Section, immunogold labeling, staining and observation
(1) The embedded block is roughly trimmed to the appropriate sample block size using a trimming machine, and the sample is trimmed to a smooth surface and a clean quadrilateral under a microscope using a blade and a glass knife.
(2) The sample was cut into ultra-thin sections of 70 nm using a diamond knife.
(3) The sections were collected using a 200 mesh carbon support membrane nickel screen.
(4) Immune gold labeling: PBS (1M, pH=7.3-7.5) was infiltrated into the sections, and the temperature was kept at room temperature for 20 minutes; blocking the non-specific protein binding sites with 1% bsa at room temperature for 20 min; 1M PBS containing 1% BSA was diluted primary antibody (CpMEDLE-2, dilution ratio 1:200) and incubated overnight at 4 ℃; samples were rinsed 4 times for 5 minutes with PBS containing 0.1% bsa 1M, pH =7.3-7.5; the secondary antibody (goat anti-rabbit with gold) was diluted with 1% bsa in 1M, pH =7.3-7.5 in PBS and incubated at 37 ℃ for 1 hour at a dilution ratio of 1:200; samples were rinsed 4 times for 5 minutes with PBS containing 0.1% bsa 1M, pH =7.3-7.5; reuse of ddH 2 O was rinsed 4 times for 5 minutes each.
(5) Heavy metal staining: uranium dyeing: 2% uranyl acetate, at 37 ℃ for 1-2 hours; ddH 2 O is bleached for 8 times, each time for 2 minutes; 10% lead citrate, and dyeing for 15 minutes at room temperature; ddH 2 O rinsing for 2 min for 10 times; and (5) airing at room temperature.
(6) The result of observation by a transmission electron microscope is shown in FIG. 3. From the figure, the cryptosporidium parvum in each intracellular period has good form and complete structure, the edges of the sodium vacuoles of the cryptosporidium parvum are smooth, and the internal structure of the cryptosporidium parvum in the intracellular period can be seen clearly. Of these, the intracellular stage of the mature type one schizont stage is observed in panel A, the intracellular stage of the small gametophyte stage is observed in panel B (indicated by the arrow), the intracellular stage of the large gametophyte stage is observed in panel C (indicated by the arrow), and the intracellular stage of the schizont stage is observed in panel D. By gold particles we see that CpMEDLE2, a protein aggregates into a gold particle circle on the membrane of the mature merozoite by Cryptosporidium parvum, and that there is also aggregation inside merozoites, and at other times there is also aggregation on the membrane but not so much. Overall, cpmele 2 protein was highly expressed during the merozoite phase of maturation, and the end result after antibody incubation was found to be distinct gold particle aggregation, but different numbers of phases were different and demonstrated good antigenicity of the samples.
Example 3
(1) Referring to the method of example 1, a slice was prepared and then immunogold-labeled: PBS (1M, pH=7.3-7.5) was infiltrated into the sections, and the temperature was kept at room temperature for 20 minutes; blocking the non-specific protein binding sites with 1% bsa at room temperature for 20 min; 1M PBS dilution of primary antibody (INS-15 polyclonal antibody containing 1% BSA, described in the literature "Xu, guo, li, et al, characial of INS-15,A Metalloprotease Potentially Involved in the Invasion of Cryptosporidium parvum[J)]2019. "dilution ratio of 1:200) was incubated overnight at 4 ℃; samples were rinsed 4 times for 5 minutes with PBS containing 0.1% bsa 1M, pH =7.3-7.5; the secondary antibody (goat anti-rabbit with gold) was diluted with 1% bsa in 1M, pH =7.3-7.5 in PBS and incubated at 37 ℃ for 1 hour at a dilution ratio of 1:200; samples were rinsed 4 times for 5 minutes with PBS containing 0.1% bsa 1M, pH =7.3-7.5; reuse of ddH 2 O was rinsed 4 times for 5 minutes each.
(2) Heavy metal staining: uranium dyeing: 2% uranyl acetate, at 37 ℃ for 1-2 hours; ddH 2 O is bleached for 8 times, each time for 2 minutes; 10% lead citrate, and dyeing for 15 minutes at room temperature; ddH 2 O rinsing for 2 min for 10 times; and (5) airing at room temperature.
(3) And (5) observing by a transmission electron microscope. The results are shown in FIG. 4, wherein, FIG. A is the intracellular stage of trophoblast stage, FIG. B is the intracellular stage of immature schizont stage, FIG. C is the intracellular stage of mature schizont stage, FIG. D is the intracellular stage of binary schizont stage, and from which we can see that each of the cryptosporidium microsporulates is well formed, the structure is intact, the edges of the cryptosporidium's sodium vacuole are smooth, the cryptosporidium internal structure of the intracellular stage can be seen, the gold particles of trophoblast stage of FIG. A are more, and the gold particles of the schizonts in FIG. B developing from trophoblast stage to schizont stage are also aggregated, but only a small amount of gold particles of the intracellular stage of mature schizont stage in FIG. C and the intracellular stage of binary schizont stage in FIG. D are aggregated on the merozoites. Taken together, it was demonstrated that the sample was well antigenic and that the protein INS-15 of Cryptosporidium parvum was expressed mainly in trophoblast.
Comparative example 1
1. Preparing a sample
(1) Two 6-8 week old female mice (IFN-. Gamma.knockout mice, purchased from the institute of medical laboratory animals, national academy of medical sciences, stock No. 002287) were prepared and starved for 12 hours before the mice were vaccinated with oocysts.
(2) Stomach of each mouse was irrigated 5X 10 5 Oocysts (diluted in PBS) were 100 μl in volume.
(3) 5-6 days after oocyst inoculation, the mice are starved again (food is taken away) when oocyst excretion reaches the peak period.
(4) Samples were taken after starvation for about 12 hours: during sampling, the sampling part is 1-2 cm part of the joint of the ileum and the cecum of the mouse in a surgical mode, the whole sampling time is controlled within 15 minutes (activity of protecting the sample), the mouse is killed by cutting the neck, the hair of the mouse is soaked by spraying 75% alcohol, the abdominal cavity is rapidly split, the whole intestinal tract is placed into precooled PBS, the ileocecal end is found, the position 1-2 cm in front of the ileocecal end is sheared, the intestinal tract is placed into new precooled PBS, and the intestinal tract is longitudinally sheared and openedRinsing in clean pre-cooled PBS 3 times, 4-5 times each, adding into pre-cooled 0.1% glutaraldehyde fixing solution (80 μL of 25% glutaraldehyde in 20000 μL of 4% paraformaldehyde) at 4deg.C, and cutting into 1-2 mm 3 Small pieces (during operation, all operations need to avoid scraping the intestinal cavity of the target intestine and damaging the cryptosporidium cyst structure to influence the result).
2. Fixing the sample
(1) The cut sample pieces were placed in a 2mL centrifuge tube, and 0.1% glutaraldehyde fixation solution (80. Mu.L of 25% glutaraldehyde in 20000. Mu.L of 4% paraformaldehyde) pre-cooled at 4℃was added to cover all tissues, and the sample was fixed at 4℃for 8 hours. After fixation the samples were rinsed 4 times with PB for 15min each.
3. Embedding a sample
(1) Gradient dehydration of ethanol: 30% ethanol at 4℃for 30 min; 50% ethanol at 4 ℃ for 1h;70%, 80% and 90% ethanol at-20 ℃ for 1 hour respectively; two times each at 100% ethanol-20℃for 1 hour.
(2) Absolute ethanol to LR-white=3:1 permeation for 2 hours at-20 ℃; absolute ethanol: LR-White = 1:1 permeation for 2 hours at-20 ℃; absolute ethanol LR-white=1:3 permeate for 2 hours at-20 ℃.
(3) LR-White was infiltrated overnight at-20 ℃.
(4) The new LR-White is changed to permeate for 24 hours at the temperature of minus 20 ℃, and then the new LR-White embedded sample is polymerized for 3 to 4 days at the temperature of minus 25 ℃ under ultraviolet (365 nm).
(5) After the sample is polymerized, the whole embedded block is taken down and put into a paper bag to be stored in a constant temperature box at 25 ℃.
4. Slice, stained slice and observation
(1) The embedded block is roughly trimmed to the appropriate sample block size using a trimming machine, and the sample is trimmed to a smooth surface and a clean quadrilateral under a microscope using a blade and a glass knife.
(2) The sample was cut into ultra-thin sections of 70 nm using a diamond knife.
(3) The sections were collected using a 200 mesh carbon support membrane nickel screen.
(4) Immune gold labeling: PBS (1M, pH)=7.3-7.5) infiltration sections, room temperature for 20 min; blocking the non-specific protein binding sites with 1% bsa at room temperature for 20 min; 1M PBS containing 1% BSA was diluted primary antibody (CpMEDLE-2, dilution ratio 1:200) and incubated overnight at 4 ℃; samples were rinsed 4 times for 5 minutes with PBS containing 0.1% bsa 1M, pH =7.3-7.5; the secondary antibody (goat anti-rabbit with gold) was diluted with 1% bsa in 1M, pH =7.3-7.5 in PBS and incubated at 37 ℃ for 1 hour at a dilution ratio of 1:200; samples were rinsed 4 times for 5 minutes with PBS containing 0.1% bsa 1M, pH =7.3-7.5; reuse of ddH 2 O was rinsed 4 times for 5 minutes each.
(5) Heavy metal staining: uranium dyeing: 2% uranyl acetate, at 37 ℃ for 1-2 hours; ddH 2 O is bleached for 8 times, each time for 2 minutes; 10% lead citrate, and dyeing for 15 minutes at room temperature; ddH 2 O rinsing for 2 min for 8 times; and (5) airing at room temperature.
(6) And (5) observing by a transmission electron microscope. As a result, as shown in FIG. 5, it was found that there were many voids (indicated by arrows) around the artemia lacunae, the internal structure of the artemia lacunae was not clear and the structure was not complete, the antigenicity was not good, and the number of gold particles accumulated in the mature schizont period (panel C) was large, but not actually present. Probably because of longer fixation time and cavitation around the worm membrane, the antibody is not hatched, the shape is bad, the structure is unclear, the number of the cryptosporidium parvum cavitation is small, and great difficulty is encountered in observation.
Comparative example 2
1. Preparing a sample
(1) Taking 1×10 7 Placing cryptosporidium parvum oocysts in a 1.5mL centrifuge tube, adding 0.5% sodium hypochlorite on ice for 10min, rinsing with PBS for 3 times, treating with 0.75% sodium taurocholate and 0.25% pancreatin (bovine pancreas for short) for a period of time at 37 ℃ (the time of storing the oocysts and the quality of the oocysts correspond to different time of removing the oocysts, fresh oocysts correspond to about 40min, and old oocysts basically complete most of the removal of the oocysts for 30 min), rinsing with PBS for 3 times, and centrifuging 13250rcf for 3 min to obtain sporozoites.
2. Fixing the sample
Adding 4% paraformaldehyde and 0.1% glutaraldehyde for fixation Fixing, fixing the sample at 4deg.C overnight, PB rinsing the sample for 5 times, centrifuging at each time for 15min at 13250rcf for 3 min, pre-embedding the sample with low melting point agarose, and cutting the sample block into 1cm pieces 3 Small blocks.
3. Embedding a sample
(1) Gradient ethanol: dehydrating 30% alcohol at 4deg.C for 30min; dehydrating 50%,70%,85%,95% alcohol at-20deg.C for 1 hr; dehydrating with 100% alcohol at-20deg.C for 1 hr;
(2) Alcohol: LR-white=1: 1, respectively penetrating for 1h at the temperature of minus 20 ℃; alcohol: LR-white=1: penetrating at-20deg.C for 1 hr;
(3) LR-White was infiltrated overnight at-20deg.C;
(4) The samples were embedded with fresh LR-White at-20℃for 8 hours, and polymerized for 3 days under ultraviolet (365 nm) at-25 ℃.
4. Slice, stained slice and observation
(1) The embedded block is roughly trimmed to the appropriate sample block size using a trimming machine, and the sample is trimmed to a smooth surface and a clean quadrilateral under a microscope using a blade and a glass knife.
(2) The sample was cut into ultra-thin sections of 70 nm using a diamond knife.
(3) The sections were collected using a 200 mesh carbon support membrane nickel screen.
(4) Immune gold labeling: PBS (1M, pH=7.3-7.5) please confirm the infiltration sections, room temperature for 20 min; blocking the non-specific protein binding sites with 1% bsa at room temperature for 20 min; 1M PBS containing 1% BSA was diluted primary antibody (CpMEDLE-2, dilution ratio 1:200) and incubated overnight at 4 ℃; samples were rinsed 4 times for 5 minutes with PBS containing 0.1% bsa 1M, pH =7.3-7.5; the secondary antibody (goat anti-rabbit with gold) was diluted with 1% bsa in 1M, pH =7.3-7.5 in PBS and incubated at 37 ℃ for 1 hour at a dilution ratio of 1:200; samples were rinsed 4 times for 5 minutes with PBS containing 0.1% bsa 1M, pH =7.3-7.5; reuse of ddH 2 O was rinsed 4 times for 5 minutes each.
(5) Heavy metal staining: uranium dyeing: 2% uranyl acetate, at 37 ℃ for 1-2 hours; ddH 2 O is bleached for 8 times, each time for 2 minutes; 10% lead citrate, and dyeing for 15 minutes at room temperature; ddH 2 O rinsing 8 times, each2 minutes; and (5) airing at room temperature.
(6) The result of observation by a transmission electron microscope is shown in FIG. 6. From the figure, the cryptosporidium parvum has almost no gold particles, the internal structure of the cryptosporidium parvum is vague, the structure is incomplete and deformed, the membrane is broken, and more holes exist, so that the aim of research cannot be achieved.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. A preparation method of an immunoelectron microscope sample for positioning specific expression proteins in cryptosporidium cells in different parasitic periods is characterized by comprising the following steps of: the method comprises the following steps:
(1) Taking HCT-8 cells as infected cells, inoculating the oocysts of the cryptosporidium subjected to the capsule removal treatment, and culturing the cells;
(2) Adding glutaraldehyde-paraformaldehyde mixed fixing solution into the cultured cells for fixing;
(3) Adding agarose with low melting point into the fixed cell sample, uniformly mixing, and pre-embedding;
(4) Centrifuging to collect a cell sample, cooling, cutting into pieces, and rinsing;
(5) Gradient dewatering the rinsed massive cell sample with ethanol, and gradient infiltration, embedding and polymerization with LR-White resin;
(6) Slicing the cell sample obtained in the step (5), performing immunogold labeling on the obtained slice, and then staining the slice with heavy metal to obtain the immunoelectron microscope sample;
the glutaraldehyde-paraformaldehyde mixed fixing solution in the step (2) is 0.1% glutaraldehyde-4% paraformaldehyde mixed fixing solution;
the specific operation of the step (2) is as follows: fixing for 1-2 h at 3-5 ℃, scraping the sheet, transferring the sheet into a new pipe, continuing to fix the sheet until the total fixing time is 4-5 h, and then discarding the fixing liquid;
the low-melting agarose in the step (3) is a low-melting agarose solution with the temperature of 38-42 ℃ and the mass concentration of 1.5%
The centrifugation conditions in the step (4) are that the temperature is 23-27 ℃, the rotating speed is 5000-7000 rpm, and the time is 3-5 minutes;
Before the centrifugation in the step (4), preheating a rotor of the centrifuge at 37-42 ℃;
the condition of the cooling treatment in the step (4) is that the temperature is 2-4 ℃ and the time is 8-12 minutes;
the gradient dehydration conditions in the step (5) are as follows: 10% ethanol at 4deg.C for 30 min, 20% ethanol at 4deg.C for 30 min, 30% ethanol at 4deg.C for 30 min, 50% ethanol at 4deg.C for 1 hr, 70% ethanol at-20deg.C for 1 hr, 80% ethanol at-20deg.C for 1 hr, 90% ethanol at-20deg.C for 1 hr, 100% ethanol at-20deg.C for 1 hr, and 100% ethanol at-20deg.C for 1 hr;
the conditions of gradient permeation in step (5) are: absolute ethyl alcohol according to the volume ratio: LR-White resin = 3:1-20 ℃ permeation 2 hours, absolute ethanol: LR-White resin = 1:1-20 ℃ permeation 2 hours, absolute ethanol: LR-White resin = 1:3-20 ℃ infiltration for 2 hours, fresh LR-White resin-20 ℃ infiltration overnight, fresh LR-White resin-20 ℃ infiltration for 24 hours;
the polymerization condition in the step (5) is that the polymerization is carried out for 3 to 4 days under the ultraviolet light with the wavelength of 315 to 370nm at the temperature of minus 25 ℃ to minus 80 ℃.
2. The method for preparing the immunoelectron microscope sample for locating specific expression proteins in cryptosporidium cells at different parasitic periods according to claim 1, wherein the method comprises the following steps:
The inoculation time in the step (1) is when the cell density of HCT-8 cells is 60% -80%;
the oocyst inoculation amount of the cryptosporidium in the step (1) is 1 multiplied by 10 according to each hole of a 12-hole plate 6 ～2×10 6 Individual oocyst/well calculation;
the stripping liquid used in the stripping treatment in the step (1) is sodium hypochlorite with the concentration of 0.4-0.6% w/v;
the condition of the capsule removing treatment in the step (1) is that the treatment is carried out on ice for 8-12 min.
3. The method for preparing the immunoelectron microscope sample for locating specific expression proteins in cryptosporidium cells at different parasitic periods according to claim 2, wherein the method comprises the following steps:
the inoculation time in the step (1) is when the cell density of HCT-8 cells is 70% -80%;
the oocyst inoculation amount of the cryptosporidium in the step (1) is 1 multiplied by 10 according to each hole of a 12-hole plate 6 Individual oocyst/well calculation;
the process of the step (1) uses sodium hypochlorite with the concentration of 0.5% w/v;
the conditions of the capsule removal treatment in the step (1) are ice treatment for 10min.
4. The method for preparing the immunoelectron microscope sample for locating specific expression proteins in cryptosporidium cells at different parasitic periods according to claim 1, wherein the method comprises the following steps:
The specific operations of the cell culture described in step (1) are: firstly culturing for 2-3 h, discarding uninjured cryptosporidium parvum oocysts, rinsing with phosphate buffer saline, adding a new culture medium, continuously culturing for 24-48 h, discarding the culture medium, and rinsing with phosphate buffer saline;
the medium used for the cell culture described in step (1) was 1640 medium containing 2% w/v fetal bovine serum.
5. The method for preparing the immunoelectron microscope sample for locating specific expression proteins in cryptosporidium cells at different parasitic periods according to claim 1, wherein the method comprises the following steps:
the thickness of the slice in step (6) is 70 nm;
the collection of the slices described in step (6) is performed by using a nickel mesh of 100 to 200 mesh carbon support film.
6. The method for preparing the immunoelectron microscope sample for locating specific expression proteins in cryptosporidium cells at different parasitic periods according to claim 1, wherein the method comprises the following steps:
the centrifugation condition in the step (4) is that the temperature is 25 ℃, the rotation speed is 6000 rpm, and the time is 3-5 minutes;
preheating the rotor of the centrifuge at 37 ℃ before the centrifugation in the step (4);
the condition of the cooling treatment in the step (4) is that the temperature is 4 ℃ and the time is 10 minutes.
7. The method for preparing the immunoelectron microscope sample for locating specific expression proteins in cryptosporidium cells at different parasitic periods according to claim 1, wherein the method comprises the following steps:
the specific operation of the immune gold labeling in the step (6) is as follows: soaking the slices with phosphate buffered saline, and standing; adding a sealing liquid for sealing; firstly adding primary anti-dilution liquid, incubating overnight at 3-5 ℃, and rinsing; adding secondary anti-dilution liquid, incubating for 1-2 h at 35-38 ℃, and rinsing;
the sealing liquid is bovine serum albumin solution with the concentration of 1-2% w/v;
the closing time is 15-25 minutes;
the primary anti-dilution liquid and the secondary anti-dilution liquid are diluted by phosphate buffer saline containing 1% bovine serum albumin and having the concentration of 1M, pH =7.3-7.5;
the dilution ratio of the primary anti-dilution liquid is 1: 100-500 parts;
the dilution ratio of the secondary anti-dilution liquid is 1: 100-500 parts;
the specific operation of heavy metal dyeing in the step (6) is as follows: firstly, dyeing in uranyl acetate at 37 ℃ for 1-2 hours, and rinsing; then dyeing in lead citrate for 15-20 minutes at room temperature, rinsing and airing at room temperature;
the uranyl acetate is a uranyl acetate solution with the concentration of 2% w/v;
The lead citrate is a lead citrate solution with the concentration of 10% w/v.
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