CN115248143A - 一种改良小鼠眼球固定液 - Google Patents
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Abstract
本发明公开了一种改良小鼠眼球固定液，所述的固定液由甲醛溶液、无水乙醇、冰醋酸和平衡盐溶液组成；将5ml甲醛溶液、35ml无水乙醇和10ml冰醋酸溶解于50ml平衡盐溶液中制得所述的固定液。采用本发明的小鼠眼球固定液对小鼠眼球固定染色的效果显著。
Description
技术领域
本发明涉及病理试剂技术领域，具体涉及一种改良小鼠眼球固定液。
背景技术
小鼠是目前眼科最常用的实验动物之一，小鼠眼球组织的病理切片常被应用于眼科相关疾病的实验研究。与其他组织器官结构不同，眼球解剖结构复杂，球内各种组织软硬度相差非常悬殊，而且眼球为腔隙结构，各组织层间连接性较差，常规固定、脱水过程易造成小鼠眼球的凹陷变形、皱缩及视网膜神经上皮层及色素上皮层之间的脱离，晶状体各结构软硬度不同，石蜡切片标本极易发生组织碎裂和细胞变形，因此眼球石蜡切片的难度较高，简单可靠的固定液及固定方法是眼球病理切片制备的关键因素之一。
发明内容
本发明要解决的技术问题是提供一种改良小鼠眼球固定液，对小鼠眼球固定染色的效果显著。
为解决上述技术问题，本发明采取如下技术方案：一种改良小鼠眼球固定液，所述的固定液由甲醛溶液、无水乙醇、冰醋酸和平衡盐溶液组成；将5ml甲醛溶液、35ml无水乙醇和10ml冰醋酸溶解于50ml平衡盐溶液中制得所述的固定液。
进一步地，所述的甲醛溶液为体积分数为40%的甲醛溶液。
进一步地，所述的平衡盐溶液按照以下质量百分比的组分组成：氯化钠0.64%、氯化钾0.070%、二水合氯化钙0.048%、六水合氯化镁0.030%、三水合醋酸钠0.39%、二水合柠檬酸钠0.17%、盐酸和/或氢氧化钠，余量为注射用水。
进一步地，采用所述的改良小鼠眼球固定液进行眼球组织制片的处理方法如下：
（1）采用改良小鼠眼球固定液室温固定24小时；
（2）将固定好的标本置于脱水盒，采用体积分数70%、80%、95%乙醇及100%乙醇逐级脱水25min，二甲苯透明2次，每次15 min，透明后的组织置于63℃液体石蜡中浸蜡3h；浸蜡后的小鼠眼球水平切面朝向包埋盒底，70℃液体石蜡包埋，蜡块冷切后，4℃保存待切；
（3）用Leica RM2135石蜡切片机切片，厚度为4μm，45℃水浴、展片，70℃烤片3h；眼球标本选取3-5张经过视***的切片依次进行二甲苯脱蜡、梯度酒精复水、水洗、苏木素染液染色10min、水洗、1%盐酸酒精分化10s、水洗返蓝、伊红染液染色1min、梯度酒精脱水、二甲苯透明10min，干燥后中性树胶封片。
进一步地，经过视***的切片进行二甲苯脱蜡2次，每次15min。
进一步地，梯度酒精复水步骤中分别采用体积分数100%乙醇、95%乙醇、80%乙醇、70%乙醇各 2 min。
进一步地，梯度酒精脱水步骤中分别采用体积分数70%、80%、95%乙醇及100%乙醇逐级脱水各1min。
本发明的有益效果为：采用本发明的小鼠眼球固定液对小鼠眼球固定染色的效果显著，小鼠眼球眼内结构形态正常、无皱缩；视网膜神经上皮与色素上皮疏松连接，脉络膜上腔可见，视网膜外核层、内核层、节细胞层等各层结构均完整、染色鲜明清晰，各层细胞排列整齐有序，无皱缩；晶状体组织结构基本完整、清晰，中央区稍碎裂；角膜各层结构整齐、层次分明。
附图说明
图1为本发明小鼠眼球石蜡切片各参数示意图。
图2为本发明四种眼球固定液固定小鼠眼球整体观。
图3为本发明四种眼球固定液对角膜形态及HE染色的比较。
图4为本发明四种眼球固定液对视***形态及HE染色的比较。
图5为本发明四种眼球固定液对视网膜形态及HE染色的比较。
图中：A0-4%甲醛固定液；B0- Davidson固定液；C0-市售眼球固定液；D0-改良小鼠眼球固定液。
具体实施方式
下面将通过具体实施方式对本发明的技术方案进行清楚、完整地描述。
本发明的一种改良小鼠眼球固定液，所述的固定液由甲醛溶液、无水乙醇、冰醋酸和平衡盐溶液组成；将5ml甲醛溶液、35ml无水乙醇和10ml冰醋酸溶解于50ml平衡盐溶液中制得本发明的固定液。本发明采用的甲醛溶液为体积分数为40%的甲醛溶液。本发明的平衡盐溶液按照以下质量百分比的组分组成：氯化钠0.64%、氯化钾0.070%、二水合氯化钙0.048%、六水合氯化镁0.030%、三水合醋酸钠0.39%、二水合柠檬酸钠0.17%、盐酸和/或氢氧化钠，余量为注射用水。
采用C57/BL6J小鼠12只24眼，将眼球随机分为4组：
A组为4%甲醛固定液；
B组为戴维森氏固定液（Davidson's固定液）；
C组为市售眼球固定液；
D组为改良小鼠眼球固定液。
比较上述本发明的固定液以及现有的A组、B组和C组固定液对小鼠眼球的固定及石蜡切片染色效果。小鼠摘除眼球后分别置于四种固定液中固定24小时，常规脱水、石蜡包埋、切片和HE染色，显微镜下观察眼球的大体结构和染色效果，比较眼球与晶状体的前后经和左右径、中央角膜及视***旁视网膜的厚度、前房深度及房角结构的固定和染色效果，具体的方法如下：
1. 材料和方法
1.1实验动物
清洁级8-10周龄雄性C57/BL6J小鼠12只，购自（南通大学实验动物中心），饲养于南通大学动物中心，饲养环境符合医学实验动物饲养要求。
1.2 主要仪器与试剂
Leica RM2135石蜡切片机、QP-B切片漂烘温控仪、DHG-9053A型电热恒温鼓风干燥箱、Motic解剖显微镜、德国徕卡Thunder显微镜。甲醛（广东光华科技股份有限公司）、无水乙醇（常试，常熟市杨园化工有限公司）、冰醋酸（上海国药集团化学试剂有限公司）、二甲苯（常试，常熟市杨园化工有限公司）、浓盐酸（上海试剂四厂昆山分厂）、苏木素染色液（北京索莱宝科技有限公司）、伊红染色液（北京索莱宝科技有限公司）、切片石蜡（上海国药集团化学试剂有限公司）、平衡盐溶液（每毫升平衡盐溶液含：氯化钠0.64%，氯化钾0.070%，二水合氯化钙0.048%，六水合氯化镁0.030%，三水合醋酸钠0.39%，二水合柠檬酸钠0.17%，盐酸和/或氢氧化钠(用于调节PH值)和注射用水）。
1.3 实验方法
1.3.1 固定液配制
A 4%甲醛固定液：体积分数为40%甲醛溶液10ml，溶解于90ml蒸馏水中。
B戴维森固定液：体积分数为40%甲醛溶液2ml，无水乙醇35ml，冰醋酸10ml，溶解于53ml蒸馏水中。
C市售眼球固定液：购自Phygene公司（PH1848）。
D改良小鼠眼球固定液：体积分数为40%甲醛溶液5ml、无水乙醇35ml、冰醋酸10ml、溶解于50ml平衡盐溶液中。
1.3.2 取材与固定
C57/BL6J小鼠颈椎脱臼法处死后，解剖显微镜下剪开球结膜，剪断球后组织和视神经，保留2mm视神经，保持眼球正常形状，PBS清洗眼球表面血液，去除结膜、筋膜及眼外肌等组织。24个眼球随机分为4组，每组6个，A组：4%甲醛固定液；B组：戴维森固定液；C组：市售眼球固定液；D组：改良小鼠眼球固定液。四种固定液室温固定24小时后，4%甲醛固定液后固定4小时，解剖显微镜下用眼科显微剪延角巩膜缘开1mm*2mm大小窗口，以利于后期脱水和石蜡浸润。
1.3.3 组织脱水与包埋
将固定好的标本置于脱水盒，体积分数70%、80%、95%乙醇及100%乙醇逐级脱水25min，二甲苯透明2次，每次15min，透明后的组织置于63℃液体石蜡中浸蜡3h。浸蜡后的小鼠眼球水平切面朝向包埋盒底，70℃液体石蜡包埋，蜡块冷切后，4℃保存待切。
1.3.4 石蜡切片与苏木素-伊红染色（HE染色）
用LeicaRM2135石蜡切片机切片，厚度为4μm，45℃水浴、展片，70℃烤片3h。每个眼球标本选取3-5张经过视***的切片进行二甲苯脱蜡（2次，每次15min），梯度酒精复水（体积分数100%乙醇、95%乙醇、80%乙醇、70%乙醇各 2min），水洗，苏木素染液染色10min，水洗，1%盐酸酒精分化10s，水洗返蓝，伊红染液染色1min，梯度酒精脱水（体积分数70%、80%、95%乙醇及100%乙醇逐级脱水各1min），二甲苯透明10min，干燥后中性树胶封片，显微镜下观察切片效果。
1.3.4 小鼠眼球石蜡切片显微拍照及相关指标测量
每个眼球标本选取经过视***切片三张，显微镜下拍照，应用莱卡Thunder显微镜拍摄全眼球图片，并测量如下指标：眼球前后径（AE）：视***巩膜顶点至角膜顶点距离；眼球左右径（FG）：经过双侧视网膜起始部位距离；晶状体前后径（CD）：晶状体前后极间的距离；晶状体左右径（HI）：晶状体赤道部的横径；前房深度（BC）：过视***中轴线上晶状体前极至角膜内皮的距离；中央角膜厚度（AB）：瞳孔区中央角膜上皮至内皮的距离；视网膜厚度(RT)：取视***旁约300μm处视网膜神经纤维层至视网膜色素上皮层的厚度。各参数测量详见图1。
2.结果
2.1 4种固定液固定后小鼠眼球标本大体形态观察
A组小鼠眼球外形和颜色无明显变化，局部稍有皱缩。B组小鼠眼球外形轻微收缩、变小，弹性较强，角膜颜色无明显变化。C组小鼠眼球明显皱缩、变形，缺乏弹性，角膜发黄、凹陷。D组小鼠眼球外形光滑饱满，未见明显变形，弹性较强，角膜颜色无变化，保持原有形态。
种固定液处理小鼠眼球标本HE染色
四种固定液固定小鼠眼球后，制备的小鼠眼球组织进行石蜡切片，每个标本选取经过视***的石蜡切片，HE染色后显微镜下观察并拍照，见表1，图2。差异如下：
A组4%甲醛固定液组小鼠眼球大体结构尚可，视网膜各层结构均能显示，但是视网膜发生严重脱离，视网膜各层排列较平整，细胞结构欠清晰，细胞间可见明显的空白裂隙。晶状体中央区出现明显组织破碎。角膜上皮各层结构较整齐，层次较分明。
B组Davidson固定液组小鼠眼球眼内结构形态基本正常，前房后房变浅或消失。视网膜轻微压缩、变薄，未脱离，视网膜分层排列平整，细胞排列整齐紧密，细胞核核膜清晰，细胞略皱缩。晶状体膨胀变大，组织结构基本完整，颜色鲜艳，对比度佳，轻微碎裂。角膜各层结构清晰、无裂隙。
C组市售眼球固定液组小鼠眼球明显变形，局部皱缩凹陷。视网膜未脱离，但是明显变薄、断裂，层次不清晰，细胞排列紊乱，细胞间界限不清晰，细胞核着色较深。晶状体变形明显，有不同程度的碎裂，染色不均匀。角膜明显凹陷、变形，细胞结构欠佳。
D组改良自制固定液组小鼠眼球眼内结构形态正常、无皱缩。视网膜神经上皮与色素上皮疏松连接，脉络膜上腔可见，视网膜外核层、内核层、节细胞层等各层结构均完整、染色鲜明清晰，各层细胞排列整齐有序，无皱缩。晶状体组织结构基本完整、清晰，中央区稍碎裂。角膜各层结构整齐、层次分明。
表1 四种固定液对小鼠眼球标本大体形态及HE染色的效果比较
| 4%甲醛 | Davidson固定液 | 市售固定液 | 改良小鼠眼球固定液 | |
| 大体结构 | 眼球轻微收缩，外形基本正常 | 眼球收缩，体积变小 | 眼球明显收缩变形，局部皱缩、凹陷 | 眼球饱满，弹性好 |
| 角膜 | 各层结构清晰，角膜基质层疏松 | 角膜变厚，略有褶皱，各层结构清晰、基质层无裂隙 | 角膜明显凹陷、变形，细胞结构欠佳 | 各层结构整齐、层次分明 |
| 前房 | 前房形态深度正常 | 前房变浅或消失 | 前房变形，深度正常 | 前房形态深度正常 |
| 晶状体 | 结构较完整清晰，中央区破碎明显，细胞排列尚可 | 膨胀变大，结构完整，中央区轻微破碎，细胞排列整齐有序 | 晶状体变形，中央部轻微碎裂，细胞排列尚可 | 结构完整，中央区轻度碎裂，细胞排列整齐有序 |
| 玻璃体腔 | 玻璃体腔存在 | 玻璃体腔变小或消失 | 玻璃体腔略变小 | 玻璃体腔正常 |
| 视网膜 | 视网膜神经上皮与色素上皮显著分离，视网膜呈V形；视网膜各层排列较平整，细胞结构欠清晰，细胞间可见明显的空白裂隙 | 视网膜神经上皮与色素上皮紧密连接，脉络膜上腔消失，未见视网膜脱离，视网膜略压缩变薄，细胞排列整齐紧密，细胞核核膜清晰，细胞略皱缩 | 视网膜神经上皮与色素上皮紧密连接，脉络膜上腔消失，未见视网膜网脱离，视网膜略压缩变薄，细胞间可见明显的空白裂隙 | 视网膜神经上皮与色素上皮疏松连接，脉络膜上腔可见，未见视网膜脱离，视网膜各层排列较平整，细胞排列整齐紧密，细胞核核膜清晰 |
| HE染色 | 颜色鲜明清晰 | 染色鲜明清晰 | 细胞核深染，颜色稍暗 | 染色鲜明清晰 |
表2 四种固定液对小鼠眼球相关参数的影响（μm）
本发明通过对四组固定液进行比较研究，结果显示本发明的改良小鼠眼球固定液是对眼球标本效果较佳的固定液，具有很好的固定效果，且对眼球大小形态的改变影响极小，HE染色鲜明清晰。
以上所述，仅是本发明的较佳实施例而已，不用于限制本发明，本领域技术人员可以在本发明的实质和保护范围内，对本发明做出各种修改或等同替换，这种修改或等同替换也应视为落在本发明技术方案的保护范围内。
Claims (7)
1.一种改良小鼠眼球固定液，其特征在于：所述的固定液由甲醛溶液、无水乙醇、冰醋酸和平衡盐溶液组成；将5ml甲醛溶液、35ml无水乙醇和10ml冰醋酸溶解于50ml平衡盐溶液中制得所述的固定液。
2.根据权利要求1所述的一种改良小鼠眼球固定液，其特征在于：所述的甲醛溶液为体积分数为40%的甲醛溶液。
3.根据权利要求1所述的一种改良小鼠眼球固定液，其特征在于：所述的平衡溶液按照以下质量百分比的组分组成：氯化钠0.64%、氯化钾0.070%、二水合氯化钙0.048%、六水合氯化镁0.030%、三水合醋酸钠0.39%、二水合柠檬酸钠0.17%、盐酸和/或氢氧化钠，余量为注射用水。
4.根据权利要求1所述的一种改良小鼠眼球固定液，其特征在于：采用所述的改良小鼠眼球固定液进行眼球组织制片的处理方法如下：
采用改良小鼠眼球固定液室温固定24小时；
将固定好的标本置于脱水盒，采用体积分数70%、80%、95%乙醇及100%乙醇逐级脱水25min，二甲苯透明2次，每次15min，透明后的组织置于63℃液体石蜡中浸蜡3h；浸蜡后的小鼠眼球水平切面朝向包埋盒底，70℃液体石蜡包埋，蜡块冷切后，4℃保存待切；
用Leica RM2135石蜡切片机切片，厚度为4μm，45℃水浴、展片，70℃烤片3h；眼球标本选取3-5张经过视***的切片依次进行二甲苯脱蜡、梯度酒精复水、水洗、苏木素染液染色10min、水洗、1%盐酸酒精分化10s、水洗返蓝、伊红染液染色1min、梯度酒精脱水、二甲苯透明10min、干燥后中性树胶封片。
5.根据权利要求4所述的一种改良小鼠眼球固定液，其特征在于：经过视***的切片进行二甲苯脱蜡2次，每次15min。
6.根据权利要求4所述的一种改良小鼠眼球固定液，其特征在于：梯度酒精复水步骤中分别采用体积分数100%乙醇、95%乙醇、80%乙醇、70%乙醇各 2min。
7.根据权利要求4所述的一种改良小鼠眼球固定液，其特征在于：梯度酒精脱水步骤中分别采用体积分数70%、80%、95%乙醇及100%乙醇逐级脱水各1min。
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