CN110646271B - 小鼠或大鼠急性胰腺炎组织石蜡切片的制备方法 - Google Patents
小鼠或大鼠急性胰腺炎组织石蜡切片的制备方法 Download PDFInfo
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Abstract
本发明提供了一种小鼠或大鼠急性胰腺炎模型胰腺组织的病理脱水和透明方法,包括如下步骤:1)将已固定的胰腺组织放入48%‑52%的乙醇浸泡3‑4h,再用68%‑72%乙醇浸泡14‑16h;2)将组织置于68%‑72%乙醇与四氢呋喃等体积混合溶液浸泡45‑55min,再将组织转移至三份四氢呋喃中分别浸泡45‑55min。本发明还提供了一种小鼠或大鼠急性胰腺炎模型胰腺组织石蜡切片和IHC样本制备方法,它是包括前述病理组织脱水方法。本发明的方法可以有效减少大、小鼠急性胰腺炎模型胰腺组织病理切片的裂痕、碎屑、褶皱,切片的完整性和连续性明显改善,具有很好的应用前景。
Description
技术领域
本发明属于动物组织病理切片制备领域,具体涉及一种急性胰腺炎小鼠或大鼠模型胰腺组织石蜡切片制备方法
背景技术
急性胰腺炎(Acute Pancreatitis,AP)是临床常见急腹症,近年来全球范围的AP发病率呈逐年上升的趋势,特别是其重症患者,常伴以系统性炎性反应综合症(SystemicInflammatory Response Syndrome,SIRS)及多器官功能障碍(Multiple OrganDysfunction Syndrom,MODS),死亡率高,住院花费大。统计数据显示,美国每年AP患者的入院人数为275,000人,与过去十年相比,增加了20%。其每年直接用于AP治疗的医疗花费高达26亿美元。我国包括北京、上海、江西在内的多地区多中心调查报告显示,各地AP发病率均呈上升趋势,以上海地区为例,AP发病率以每年5.1%的速率递增,每位AP患者的住院花费约3万元人民币。然而,目前全球范围内尚缺乏针对AP的特异性治疗措施,其发病机制始终不清。因此针对AP的诊疗及机制研究一直是消化、外科及重症医学多学科临床工作及基础研究的重点、热点。
统计数据显示,过去五年时间,pubmed上发表的AP相关研究5655篇,国内主要期刊平台,包括知网、万方及维普在内,可检索到急性胰腺炎相关研究共计37438篇。在这些众多的AP相关研究中,大鼠、小鼠因其价格相对经济,繁殖率高,便于进行手术操作及基因改造等优点,是目前AP研究中使用频率(90%以上)最高实验动物模型。苏木精—伊红染色法(hematoxylin-eosin staining,HE)和免疫组织化学(免疫组化,Immunohistochemistry,IHC)是生物医学研究及病理诊断中最基本的方法。HE染色法是利用组织或细胞的不同成分对苏木精和伊红的亲和力及染色性质不同,以显示组织病变的形态结构特点。IHC是通过组织原位抗原抗体反应和组织化学的呈色反应,对靶抗原进行定性、定位、定量的检测。对小鼠进行AP造模,取其胰腺组织进行HE及IHC染色及检测,是AP研究中不可或缺的技术手段。HE和IHC是基于石蜡切片进行的进一步染色及检测,石蜡切片的制作过程包括固定、脱水、透明、浸蜡、包埋、切片、展片和粘片、烤片等环节,对于IHC,还包括抗原修复。
上述每个环节的实验条件因组织类型及模型种类不同而有所不同。其中脱水是指脱去组织内的水分,即借助某些溶媒置换组织内水分的过程。一般经过固定的组织内含有较多水分,而水是不能与蜡相混合的,因此在浸蜡之前必须充分脱去组织块中的水分。脱水环节条件把握在整个制片过程中非常重要,若组织脱水条件不当,会直接导致后续切片产生裂纹、褶皱、碎屑,出现展片时间长、废片率高等一系列问题。通常,组织固定后经乙醇、二甲苯脱水制成石蜡切片,是常规采用的流程。然而,与肝、肾等实质器官不同,小鼠胰腺组织结构松散,特别是造模后的AP小鼠胰腺组织,以严重的水肿、坏死及炎性细胞浸润为特征,与正常胰腺组织及其它脏器相比较,具有含水量显著升高,同时伴有细胞间间隙增大、柔软性强的特点,按照普通样本脱水及制片流程处理,易造成组织脆硬、收缩,后期切片出现碎屑、裂痕的比例高等问题。此外,在应用于IHC进行切片修复时,造模后的AP胰腺组织因小叶间间隙增宽,组织结构疏松,浸润的炎性细胞和管状结构(胰管及血管内皮)因无支撑结构或支撑弱,常出现脱片现象,以此造成重要结构及信息的丢失,严重妨碍了AP科学研究的观察及检测。
从技术流程上来看,常规石蜡切片制作过程中,脱水步骤设置4-6个乙醇浓度梯度,每个梯度维持30min-2h,有些梯度还需重复进行,此外,还需再进行两次二甲苯的透明步骤,总体上来讲操作较为繁复。
发明内容
为了解决上述问题,本发明提供了一种专门针对急性胰腺炎大、小鼠模型胰腺组织的脱水和透明相结合的方法,以及对应的石蜡切片的制作方法、免疫组化样品制备方法。
本发明的技术方案包括:
一种急性胰腺炎小鼠或大鼠模型胰腺组织的病理脱水和透明方法,包括如下步骤:
1)将已固定的胰腺组织放入48%-52%的乙醇浸泡3-4h,再用68%-72%乙醇浸泡14-16h;
2)将组织置于68%-72%乙醇与四氢呋喃等体积混合溶液浸泡45-55min,再在四氢呋喃中浸泡3次,每次45-55min。
如前述的脱水和透明方法,所述步骤1)是将已固定的胰腺组织放入50%的乙醇浸泡3-4h,再用70%乙醇浸泡14-16h。
如前述的脱水和透明方法,所述步骤2)中的混合溶液是70%乙醇与四氢呋喃等体积混合溶液;
和/或,混合溶液浸泡时间是50min;
和/或,四氢呋喃中的浸泡时间是50min。
一种急性胰腺炎小鼠或大鼠模型胰腺组织石蜡切片制备方法,其特征在于,它包括如前所述的脱水和透明方法。
如前述的急性胰腺炎小鼠或大鼠模型胰腺组织石蜡切片制备方法,包括:
1)固定:固定胰腺组织36-48小时;
2)脱水和透明:使用权利要求1~3任一所述方法进行脱水和透明;
3)包埋和切片;
4)展片和烤片:将切片在47~50℃水中展平,使用玻片裱片并于38-42℃烘箱烤干。
进一步地,步骤4)中水温为50℃,烘箱中的温度为40℃。
进一步地,步骤1)是使用4%甲醛溶液进行固定。
一种急性胰腺炎小鼠或大鼠模型胰腺组织免疫组化样品制备方法,它是在前述石蜡切片制备方法的基础上,加上如下步骤:
5)在58-62℃烤片1h;
6)切片浸入抗原修复液,经4次微波炉加热修复;第一次为加热到58-62℃,维持7min;待冷却至25-35℃后,再三次加热到38-42℃,各维持6min;每次需待修复液待冷却至25-35℃后再进行下一次加热。
抗原修复液是柠檬酸钠抗原修复液或者Tris-EDTA抗原修复液。
进一步地,步骤5)为在60℃烤片1h。
进一步地,步骤6)中第一次加热到60℃,维持7min;后三次加热40℃,各维持6min。
本发明具有如下有益效果:
本发明的方法,充分考虑了急性胰腺炎组织的柔软性强、含水量高的特点,从较低浓度(50%)的酒精开始脱水,再使用70%酒精脱水,使脱水过程温和,能减小组织形变,减少后期组织裂痕,可加快后期的展片速度。此外,发明人通过经验摸索,调整了脱水时间,延长70%酒精浸泡时间,是为了让脱水剂充分浸入组织内部,充分提高后期切片的完整性,从而改善后续切片连续性差,降低切片碎屑、裂痕比例。另一方面,使用70%酒精与四氢呋喃的混合溶液同时兼顾脱水和透明,简化操作流程。
相比传统方法,本发明的方法从50%浓度酒精开始脱水,使脱水过程温和,能减小组织形变,减少后期组织裂痕,可加快后期的展片速度。同时减少了脱水所用酒精的梯度数量,能减少实验人员的操作步骤,节省人力成本。此外,使用四氢呋喃替换常规方案中的二甲苯,兼顾脱水和透明的双重作用,能够改善组织易碎、展片慢、褶皱多的问题。
相比郑翔等的方法,最主要的是延长了70%酒精浸泡时间(郑翔方案2小时,本方案14-16小时)。延长70%酒精浸泡时间,是为了让脱水剂充分浸入组织内部,提高后期切片的完整性,改善切片连续性差,降低切片碎屑、裂痕比例。此外,本发明通过经验摸索减少了四氢呋喃透明所花费的时间,是事先未预料到的。本领域技术人员知晓,透明步骤的目的是置换掉脱水步骤进入组织的酒精,而本发明方法相比郑翔等的方法,脱水用的酒精浓度一致,但脱水时间更长,按常理来说需要更长的透明时间来置换掉进入组织的酒精。但是本发明从第三缸到第六缸的4个四氢呋喃处理步骤每步缩短了10min,还达到了更好的切片制备效果。
大鼠和小鼠的生理特征接近,理论上本发明的脱水和透明方法、石蜡切片制备方法以及免疫组化样品的制备方法同样适用于大鼠急性胰腺炎组织。
总之,本发明的方法可以有效减少切片的裂痕、碎屑、褶皱,提升切片的完整性和连续性,本发明的石蜡切片方法可以减少乙醇脱水的步骤,并减少透明所花时间;具有很好的应用前景。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过具体实施方式对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1:切片的裂片情况对比。A:常规方案;B:郑翔等方案;C:本方案。400×放大倍数,标尺(scale bar):50μm。
图2:切片的叠片情况对比。A:常规方案;B:郑翔等方案;C:本方案。100×放大倍数,标尺(scale bar):200μm。
具体实施方式
实施例1胰腺炎小鼠模型胰腺组织石蜡切片制备
1.方法
(1)取材
使用眼科剪沿十二指肠取下胰腺炎小鼠模型的胰腺组织。
造模方式为:选用C57/BL6小鼠,6-8周龄,体重20-25g。经腹腔注射雨蛙素50μg/kg,每小时注射1次,共7次,最后一次合并注射15mg/kg的脂多糖。
(2)固定
使用固定液固定36-48h,固定液的配方是:甲醛溶液(含40%甲醛)100ml,十二水磷酸氢二钠13g,二水磷酸二氢钠4g,蒸馏水900ml。
(3)脱水和透明
使用50%酒精浸泡3-4小时,70%酒精浸泡14-16h。
然后置于70%乙醇与四氢呋喃等体积混合溶液浸泡50min,再将组织捞出,依次在三份四氢呋喃中分别浸泡50min。此脱水过程还具有透明的作用。
(4)包埋和切片
将透明后的组织与60℃下浸蜡3h以上,随后用热融的石蜡包埋,冷却。石蜡块凝固好后即可使用切片机切片,得石蜡切片。
(5)展片和烤片
将得到的石蜡切片在50℃水中展平(展片),裱片并烤干;烤片条件为:40℃烤片14-16h。
实施例2胰腺炎小鼠模型胰腺组织免疫组化样品的制备
步骤(1)~(5),同实施例1。
(6)额外烤片
在60℃烤片1h。
(7)抗原修复
切片浸入抗原修复液,经4次微波炉加热修复;第一次为加热到60℃,维持7min;后三次加热到40℃,各维持6min;每次加热后需冷却至室温再进行下一步加热。
抗原修复液可以是柠檬酸钠抗原修复液,也可以是Tris-EDTA抗原修复液;2种修复液的选择视情况而定:前者主要适用于膜和胞浆蛋白的检测,后者主要适用于核内蛋白的检测。
I.柠檬酸钠抗原修复液的一般配方为:
A液:柠檬酸溶液(0.1mol/L)柠檬酸21.01g加双蒸水1L;
B液:柠檬酸钠(0.1mol/L)柠檬酸钠29.41g加双蒸水1L;
抗原修复液:A液9mL+B液41mL+双蒸水450mL。
II.Tris-EDTA抗原修复液的一般配方为:
Tris 30.27g;
EDTA(乙二胺四乙酸) 1.461g;
蒸馏水 定容至500ml;
用前稀释10倍。
上述两种修复液均有对应商品,亦可用商品替代。
实施例3胰腺组织免疫组化检测方法
将实施例2所制备得到的免疫组化样品按如下步骤检测。
1.封闭
3%过氧化氢避光孵育15分钟——PBS清洗三次,每次五分钟
2.抗原抗体反应
1)按照不同目的蛋白抗体说明书稀释一抗,每个样本滴加约50ul一抗稀释液,4℃孵育过夜;
2)第二天从冰箱里取出样本后,在室温环境中复温1小时。
3)PBS洗片3次,每次5min。按照一抗种属选择对应二抗,二抗37℃孵育30分钟;
4)PBS洗片3次,每次5min。
5)滴入DAB显色液,显微镜下观察,显色。
3.封片
在组织正中滴入约20-50ul中性树胶封片剂,盖上盖玻片,避免气泡。
对比例1小鼠造模前、后胰腺组织及肝、肾组织含水量比较
切取正常小鼠及模型小鼠胰腺、肝、肾等组织于电子天平称组织湿重,铝箔包裹于70℃烤箱烘烤72小时,再用电子天平称取组织干重。计算组织含水量(反应水肿程度):
含水量=(组织湿重-组织干重)/组织湿重×100%
结果发现,小鼠急性胰腺炎组织的含水量高达82.16%,相比正常胰腺组织含水量明显升高,且明显高于正常肝组织和正常肾组织(如表1)。
表1小鼠造模前、后胰腺组织及肝、肾组织含水量比较
*P<0.05
对比例2本发明的石蜡切片与其他2种石蜡切片的脱水(含透明)环节对比及效果对比
1.脱水和透明步骤细节对比
见表2。
表2三种石蜡切片的脱水(含透明)环节的对比
2.切片质量对比
取100例AP造模后的小鼠胰腺组织,均分为三部分,分别使用本方案、常规方案及郑翔等的方案进行脱水和透明对其进行样本处理及制片(后续步骤同实施例1,并进行HE染色),对切片的裂痕、连续性、碎屑、褶皱及切片完整性等5个指标进行评价。
采用SPSS16.0软件进行统计学处理,实验结果采用Pearson x2检验计数资料。结果见表3。
表3各组病理切片质量指标比较
*P<0.05
本发明的切片制备方法还能减少裂片(图1)和叠片(图2)情况。
综上,本发明的脱水和透明方法,适合小鼠急性胰腺炎组织这类含水量高的组织,组织脱水过程温和且彻底;因此后续步骤切片的裂痕、碎屑、褶皱减少,完整性和连续性得到极大的改善。本发明的方法还可以减少乙醇脱水的步骤,并减少透明所花时间。本发明的方法具有十分良好的应用价值。
Claims (8)
1.一种急性胰腺炎小鼠或大鼠模型胰腺组织的病理脱水和透明方法,其特征在于,包括如下步骤:
1)将已固定的胰腺组织放入50%的乙醇浸泡3-4h,再用70%乙醇浸泡14-16h;
2)将组织置于70%乙醇与四氢呋喃等体积混合溶液浸泡50min,再在四氢呋喃中浸泡3次,每次50min。
2.一种急性胰腺炎小鼠或大鼠模型胰腺组织石蜡切片制备方法,其特征在于,它包括如权利要求1所述的脱水和透明方法。
3.如权利要求2所述的石蜡切片制备方法,其特征在于,包括:
1)固定:固定胰腺组织36-48小时;
2)脱水和透明:使用权利要求1所述方法进行脱水和透明;
3)包埋和切片;
4)展片和烤片:将切片在47~50℃水中展平,使用玻片裱片并于38-42℃烘箱烤干。
4.如权利要求3所述的石蜡切片制备方法,其特征在于,步骤4)中水温为50℃,烘箱中的温度为40℃。
5.如权利要求3或4所述的石蜡切片制备方法,其特征在于,步骤1)是使用4%甲醛溶液进行固定。
6.一种小鼠或大鼠急性胰腺炎模型胰腺组织免疫组化样品制备方法,其特征在于,它是在权利要求2~5任一所述方法的基础上,加上如下步骤:
5)在58-62℃烤片1h;
6)切片浸入抗原修复液,经4次微波炉加热修复;第一次为加热到58-62℃,维持7min;
待冷却至25-35℃后,再三次加热到38-42℃,各维持6min;每次需待修复液待冷却至25-35℃后再进行下一次加热;
抗原修复液是柠檬酸钠抗原修复液或者Tris-EDTA抗原修复液。
7.如权利要求6所述的组织免疫组化样品制备方法,其特征在于,步骤5)为在60℃烤片1h。
8.如权利要求6所述的组织免疫组化样品制备方法,其特征在于,步骤6)中第一次加热到60℃,维持7min;后三次加热40℃,各维持6min。
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