CN116106108B - Cell wax block and embedding method for frozen sections of small number of cells - Google Patents

Abstract

The invention relates to the technical field of biomedical treatment, in particular to a method for embedding cell wax blocks and a small amount of cell frozen sections, which comprises the following steps that (S.3) dehydrated cells are subjected to small-volume pre-embedding through a colored embedding agent, and the cells are solidified to obtain pre-embedding blocks containing the cells; and (S.4) transferring the pre-embedded block into a colorless embedding agent, and solidifying again after the pre-embedded block is completely melted to obtain the embedded block containing cells. The invention marks the embedded cell mass by using the colored embedding agent, is convenient for judging whether to start collecting the slice and judging whether to completely cut the embedded object, and simultaneously uses the pre-embedding mode to lead the embedded cell mass to be more concentrated, thereby ensuring that each slice has a plurality of target slices.

Description

Cell wax block and embedding method for frozen sections of small number of cells

Technical Field

The invention relates to the technical field of biomedical treatment, in particular to a cell wax block and a method for embedding a small number of cells in frozen sections.

Background

Immunofluorescent staining diagnosis is a diagnosis method for pathological conditions by using immunofluorescent staining and a microscope, is commonly used for checking various specimens of patients suffering from immune diseases, and is mainly used for diagnosing diseases by observing the high specificity of antigen-antibody reaction through fluorescent labeling of antigen-antibody.

Islets refer to the endocrine portion of the pancreas, which is a mass of cells of varying size and shape, scattered throughout the pancreas, and the hormone insulin produced by the islets controls carbohydrate metabolism, typically 50-300um in diameter. Islet-like cells refer to cell masses obtained from human pluripotent stem cells by means of directed induction differentiation or transdifferentiation, which are structurally similar to islets and functionally equivalent to human pluripotent stem cells, and whose diameters can be artificially regulated, but do not exceed 300um. Immunofluorescent staining of islet or islet-like cells is a relatively common detection procedure that first requires that the sample be prepared into a slice.

Methods of making sections generally include both paraffin sections and frozen sections. Wherein paraffin sections can better maintain the morphological structure of tissue cells and are easy to store inHowever, paraffin sections are complicated in preparation process, and long-time formalin fixation and ethanol dehydration in the preparation process can lead to antigen blocking, so that the staining effect of the paraffin sections on the paraffin sections is often poor in later repair. Meanwhile, the paraffin section sample preparation process is complex, the paraffin section sample preparation process is generally applicable to volume through the steps of multi-step dehydration, paraffin dipping and the like after fixation>1 mm ³ While islets or islet-like cells have a diameter of less than 300 a um a are extremely prone to loss during paraffin section preparation, paraffin sections are not suitable for this type of cells.

Frozen section (frozen section) is a method of rapidly cooling tissue to a certain hardness under low temperature conditions and then slicing (for example, a method of producing an improved frozen section of an eyeball of a mouse as shown in application number CN 202010143762.2). Because the sample preparation process is in a low-temperature state, the fixing liquid is mild, and the fixing time is short, so that the protein cannot be excessively crosslinked, and the immunofluorescence staining effect is better, and therefore, the immunofluorescence staining is generally carried out by adopting a frozen section sample.

However, the applicant has found the following problems in applying frozen sections to immunofluorescent staining of islets or islet-like cells: (1) Since the islets or islet-like cell masses are small and not easily observed, it is not easy to determine whether islets or islet-like cell masses have been cut or not at the time of slicing; (2) When the islet or islet-like cell mass sample is less, the sample is dispersed after the islet or islet-like cell mass sample is embedded, and only a small amount of islet or islet-like cells are even absent on a single slice, so that the later staining analysis is not facilitated. Thus, there is no prior art in which frozen sections are applied to islets or islet-like cell clusters.

Disclosure of Invention

The invention provides a method for embedding a small amount of cells in frozen sections, which aims to solve the problems that the cell clusters are not easy to find in the frozen sections in the prior art, the cell clusters are scattered, and the cell clusters on a single section are less or even not.

In order to achieve the aim of the invention, the invention is realized by the following technical scheme:

in a first aspect, the present invention provides a method for embedding a small number of cells in frozen sections,

comprises the following steps

(S.3) carrying out small-volume pre-embedding on the dehydrated cells through a colored embedding agent, and solidifying to obtain a pre-embedding block containing the cells;

and (S.4) transferring the pre-embedded block into a colorless embedding agent, and solidifying again after the pre-embedded block is completely melted to obtain the cell wax block containing cells.

Frozen sections of the prior art are typically prepared by removing dehydrated cells, transferring the cells directly to an OCT embedding medium, and mixing the cells with the embedding medium to solidify the sections. However, the applicant of the present invention found that when these dehydrated cells are dispersed in an embedding medium, the cells are fused together with the embedding medium, and therefore it is difficult to determine whether or not cells are present in the slice from the appearance of the slice. Meanwhile, even if cells are present in a slice, there is a problem that the number of cells present on a single slice is small. Furthermore, as described in the background art, during the observation of these sections, individual cells are difficult to observe even in sections in which they are present, because of their small volume (e.g., islet or islet-like cells as described in the background art).

The invention creatively uses a two-step embedding method, and solves the problems that cell clusters are not easy to find in the slicing process and the cell clusters are less or even not on a single slice. Firstly, the invention does not directly transfer the conventional cells into the conventional OCT embedding agent after the conventional cells are dehydrated, but firstly adopts a small-volume pre-embedding method to pre-embed the cells through the colored embedding agent, thereby obtaining the pre-embedded block. In the pre-embedding blocks, because of the small volume, the density of cells embedded in the pre-embedding blocks is large, and when the pre-embedding blocks are cut in the process of preparing frozen slices, the number of cells in the slice layers on the pre-embedding blocks is higher than that of the cells in the slices prepared by the traditional embedding means.

However, the small-volume pre-embedded block is difficult to slice by conventional slicing equipment because of its small volume. Thus, the present invention also performs a secondary embedding of this pre-embedded block after the small-volume pre-embedding. The embedding agent selected in the secondary embedding process is a colorless embedding agent, and at the same time, because the viscosity of the embedding agent (including the colored embedding agent in the step (s.3) and the colorless embedding agent in the step (s.4)) is relatively large, color diffusion does not occur between the embedding agents of two different colors, and cells wrapped in the colored embedding agent are also confined in the colored embedding agent and are not transferred into the colorless embedding agent, so that the density of the cells in the colored embedding agent can be kept from being reduced after solidification. When the obtained pellet is sliced, the colored embedding medium is cut, which indicates that the target cells have been cut. At this time, only the colored region in the middle of the slice is observed, so that a plurality of target sections can be ensured for each slice, and the smooth implementation of final immunofluorescence staining is facilitated.

Preferably, the cells include any one of islet cells, islet-like cell clusters, exfoliated cells, or single cells.

The freezing section embedding method overcomes the severe requirements on cells in the section preparation process in the prior art, has no excessive limitation on the size and the number of the cells, can well embed the cells with small diameter and smaller sample amount, ensures that the prepared section is easier to observe, and simultaneously has more cell number on a single section.

Preferably, said step (s.3) is carried out in a centrifugal column;

and further comprises the step of centrifuging the column containing the dehydrated cells to remove the dehydrating agent.

The applicant has noted that when the target cells are islet or islet-like cell masses, they are highly adherent to the surface of the tip of the pipette due to their high viscosity, thus resulting in loss of cells during each pipetting, especially during removal of the dewatering agent used for dewatering the cells. This is especially true for small amounts of islet or islet-like cell mass. In addition, the dehydrating agent in the prior art is very difficult to completely absorb through the pipettor, so that a part of dehydrating agent still remains after target cells are dehydrated, and ice crystals are formed after the part of residual dehydrating agent is frozen, and the ice crystals are easy to wrinkle or fracture during slicing, so that the quality of the sliced slices is poor.

In order to solve the problem, the step (S.3) is specially carried out on the centrifugal column, and meanwhile, the step of centrifugally removing the dehydrating agent from dehydrated cells is added.

In addition, because the target cells are completely gathered at the bottom of the centrifugal column after centrifugation, a small amount of colored embedding agent is added into the centrifugal column at this time to embed all the target cells, so that the cell density in the obtained pre-embedded block is greatly improved.

Preferably, the step (S.3) comprises at least 5 cells or cell clusters per microliter of colored embedding medium.

The addition amount of the colored embedding agent can be regulated and controlled according to the number of target cells or cell clusters at the bottom of the centrifugal column, so that the cell density in the final pre-embedded block is ensured to be enough to enable a plurality of target sections to be observed in each slice.

Preferably, the curing conditions in the step (s.3) and the step (s.4) are as follows: the embedding medium was placed over but not in contact with liquid nitrogen until the embedding medium was completely solidified.

Preferably, the specific steps in the step (s.4) are as follows: transferring the solidified pre-embedding block into a colorless embedding agent, completely immersing the pre-embedding block into the colorless embedding agent and placing the pre-embedding block in the center position, and solidifying the pre-embedding block again after the pre-embedding block is completely melted, so as to obtain an embedding block containing cells, and then slicing the embedding block or placing the embedding block in a condition of-80 ℃ for preservation.

Preferably, the method further comprises the following steps:

(S.1) transferring the cells into a centrifugal column, and fixing the cells in the centrifugal column by a fixing solution;

(S.2) adding a dehydrating agent to the cell-immobilized centrifugal column to dehydrate the cells.

Preferably, the step (s.1) specifically includes the following steps:

(S.1.1) collecting the cells into a centrifugal column;

(S.1.2) removing the cell culture broth by centrifugation;

(S.1.3) washing the cells from which the cell culture solution is removed to remove the residual cell culture solution;

(S.1.4) adding a fixing solution into the centrifugal column, so that cells are fixed in the centrifugal column;

(S.1.5) washing the cells immobilized in the column to remove the residual fixative.

Preferably, the fixing solution is any one of paraformaldehyde solution, formaldehyde, glutaraldehyde, ethanol and acetone.

Preferably, the step (s.2) further includes a step of judging whether the dehydration of the cells is completed, and the method specifically includes:

after the dehydration step is finished, the dehydrating agent in the centrifugal column is removed by centrifugation, then the dehydrating agent is added again, the dehydration is finished when the cell mass can be deposited at the bottom, and the incomplete dehydration is indicated if the cell mass cannot be deposited at the bottom.

Preferably, the dehydrating agent comprises any one of sucrose solution, ethanol, acetone, n-butanol, tert-butanol, cyclohexanone and pinoresinol.

In a second aspect, the invention also provides a cellular wax block obtainable by said method,

comprises a colorless embedding region; the method comprises the steps of,

a colored embedding region located inside the colorless embedding region;

cells are uniformly dispersed in the colored embedding region.

Therefore, the invention has the following beneficial effects:

(1) The colored embedding agent is used for marking the embedded cell mass, so that whether the section is collected or not and whether the embedded object is completely cut or not can be judged conveniently;

(2) The embedded cell clusters are concentrated by using a pre-embedding mode, so that each slice can be ensured to have a plurality of target sections;

(3) The liquid is removed by using a centrifugal filtration mode, so that the cell mass is ensured to be completely free from loss in the whole process;

(4) The liquid can be completely removed by centrifugal filtration, and no ice crystal is formed in the sample preparation process.

Drawings

FIG. 1 is a schematic diagram of a method for embedding a small number of frozen sections of cells according to the present invention.

FIG. 2 is a graph showing the practical effect of the method for embedding a small number of frozen sections of cells according to example 1 of the present invention.

FIG. 3 is a schematic structural diagram of the slice prepared in example 1.

FIG. 4 is the number of cell clusters per slice after the same number of islet-like cell clusters were embedded in the treatment according to both of the two technical solutions described in example 1 and comparative example 1.

FIG. 5 is a graph showing the effect of single cell embedding and slicing in example 2.

Wherein: colorless embedding region 10, colored embedding region 20, cells 30.

Detailed Description

The invention is further described below with reference to the drawings and specific examples. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.

Example 1

As shown in fig. 1-2, the method for embedding a small quantity of frozen sections of cells in the invention specifically comprises the following steps:

(S.1) cell immobilization:

(s.1.1) transferring islet-like cell mass obtained by directional induction differentiation or by transdifferentiation of islet-like cell mass obtained by isolating islet or human pluripotent stem cells in pancreatic tissue into a centrifugal column (Thermo, 87767);

(S.1.2) placing the centrifugal column on a 2ml round bottom uncovered centrifugal tube, and centrifuging 100g for 1 minute to remove the cell culture solution;

(S.1.3) adding 500ul of PBS to the column, centrifuging 100g for 1 min to remove the PBS;

(S.1.4) into the centrifugal column, 500ul 4% Paraformaldehyde (PFA) solution was added, the mixture was fixed at 4℃for 30 minutes, and 100g was centrifuged for 1 minute to remove the fixing solution;

(S.1.5) to the centrifugal column in 500ul PBS,100g centrifugal 1 minutes to remove PBS, repeated 3 times.

(S.2) dehydration of cells:

(S.2.1) adding 500ul of 30% sucrose solution into the centrifugal column, and dehydrating in a refrigerator at 4 ℃ for 24 hours;

(S.2.2) centrifuging 100g for 1 min to remove the sucrose solution, adding 500ul of 30% sucrose solution into the centrifugal column, if the cell mass can settle to the bottom, indicating that the dehydration is completed, if the cell mass cannot settle to the bottom, indicating that the dehydration is not complete, and continuing the dehydration until the cell mass can settle to the bottom.

(S.3) cell pre-embedding:

(S.3.1) centrifuging 300g for 3 minutes, completely removing the sucrose solution, after the liquid is removed, dripping a small amount of colored embedding agent (5 cell mass per microliter of embedding agent, adjusting the volume of the embedding agent according to the number of islets or islet-like cell mass) (Lecia, 3801480) into the bottom of the centrifugal column, and fully and uniformly mixing the cell mass and the embedding agent;

(S.3.2) the column was placed over but not in contact with liquid nitrogen until the embedding medium was completely solidified and the embedded block was removed from the bottom of the column with forceps.

(S.4) cell embedding:

(s.4.1) transferring the solidified pre-embedding block into a colorless embedding agent (SAKURA, 4583), completely immersing the pre-embedding block into the colorless embedding agent and placing the pre-embedding block in a central position, and placing an embedding box on liquid nitrogen but not contacting the liquid nitrogen for solidification after the pre-embedding block is completely melted;

(S.4.2) slicing the cell wax block after complete solidification or preserving at-80 ℃.

As shown in fig. 3, the cut surface structure of the slice obtained by the above method is shown in fig. 3, which includes a colorless embedding region 10 obtained by solidification of a colorless embedding agent; the method comprises the steps of,

a colored embedding region 20 located inside the colorless embedding region 10 and obtained after solidification of the colored embedding agent;

cells 30 are uniformly dispersed in the colored embedding region 20.

Comparative example 1

In order to compare the effect of the present invention with that of the prior art frozen section embedding method, a comparison test was purposely performed according to the prior art.

(1) Collecting islet or islet-like cell mass into a 1.5ml centrifuge tube;

(2) Centrifuging by a centrifuge (300 g,3 min) to remove supernatant;

(3) 200 ul of 4% PFA ice bath was added for fixation for 30 min (this step can be performed in staining);

(4) Centrifuging by a centrifuge (300 g,3 min) to remove PFA;

(5) Adding 200 ul PBS, soaking for 3 min, centrifuging (300 g,3 min), and washing for 3 times;

(6) After PBS is removed, 1.3 ml of 30% sucrose is added, and the mixture is dehydrated at 4 ℃ until the cell mass is settled to the bottom of a tube (generally, the treatment is carried out for 24 hours);

(7) Sucking out sucrose, taking out cells from a 1.5ml centrifuge tube, and transferring the cells into an OCT embedding agent;

(8) Placing above liquid nitrogen (without contacting with liquid nitrogen), slicing the cell wax block after completely solidifying or preserving at-80deg.C.

[ slice Performance test ]

The cell mass on the sections prepared in example 1 and comparative example 1 was observed, and the number of cell masses on each number of sections was recorded. As can be seen from FIG. 4, the method for embedding frozen sections can effectively increase the number of cell clusters on each section, thereby effectively improving the detection accuracy in the immunofluorescent staining process of islet or islet-like cells.

Example 2

Example 2 differs from example 1 in that islet-like cells were replaced with single cells, while in order to fit single cells, the centrifuge column was replaced with a smaller bore centrifuge column (Pierce ™ C18 89870 in this example).

Fig. 5 is a graph showing the effect of single cell embedding in example 2, and the method for embedding frozen sections of the present invention can effectively increase the number of cell clusters on each section for single cells with smaller volume, thereby effectively improving the detection accuracy in the immunofluorescence staining process of islets or islet-like cells.

Claims (5)

1. A method for embedding a small number of cells in frozen sections, comprising the steps of:

(S.1) transferring the cells into a centrifugal column, and fixing the cells in the centrifugal column by a fixing solution;

the step (S.1) specifically comprises the following steps:

(S.1.1) collecting the cells into a centrifugal column;

(S.1.2) removing the cell culture broth by centrifugation;

(S.1.3) washing the cells from which the cell culture solution is removed, and centrifuging to remove the residual cell culture solution;

(S.1.4) adding a fixing solution into the centrifugal column, so that cells are fixed in the centrifugal column;

(S.1.5) washing the cells immobilized in the centrifugal column, and centrifuging to remove residual immobilization liquid;

(S.2) adding a dehydrating agent to the cell-immobilized centrifugal column to dehydrate the cells;

(S.3) centrifuging a centrifugal column containing dehydrated cells to remove a dehydrating agent, pre-embedding the dehydrated cells in a small volume through a colored embedding agent, and solidifying to obtain a pre-embedded block containing the cells;

(S.4) transferring the solidified pre-embedding blocks into a colorless embedding agent, enabling the pre-embedding blocks to be completely immersed into the colorless embedding agent and placed in a central position, solidifying the pre-embedding blocks again after the pre-embedding blocks are completely melted, thus obtaining embedded blocks containing cells, and then slicing the embedded blocks or preserving the embedded blocks at the temperature of minus 80 ℃;

the cells include any one of islet cells, islet-like cell clusters, exfoliated cells, or single cells.

2. A method for embedding a small number of cells in frozen section according to claim 1,

at least 5 cells or cell clusters per microliter of colored embedding agent are included in step (s.3).

3. A method for embedding a small number of cells in frozen section according to claim 1,

the fixing liquid is any one of paraformaldehyde solution, formaldehyde, glutaraldehyde, ethanol and acetone.

4. A method for embedding a small number of cells in frozen section according to claim 1,

the step (s.2) further includes a step of judging whether the dehydration of the cells is completed, and specifically includes:

after the dehydration step is finished, the dehydrating agent in the centrifugal column is removed by centrifugation, then the dehydrating agent is added again, the dehydration is finished when the cell mass can be deposited at the bottom, and the incomplete dehydration is indicated if the cell mass cannot be deposited at the bottom.

5. A method for embedding a small number of cells in frozen section according to claim 1 or 4,

the dehydrating agent comprises any one of sucrose solution, ethanol, acetone, n-butanol, tertiary butanol, cyclohexanone and pinoresinol.