CN108362538A - 一种皮肤病理切片的制作方法 - Google Patents
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Abstract
本发明公开了一种皮肤病理切片的制作方法,该方法通过活体取材,离体皮肤组织固定,离体组织脱水,浸蜡、包埋,切片,染色制得皮肤病理切片,将动物皮肤、皮下组织的取材、固定和包埋方法规范化,解决了皮肤病理标本与活体组织形态一致性差、病理图片信息不完整的问题,还能在不显著增加操作难度的前提下,为皮肤免疫组织化学、免疫荧光染色提供自身对照。
Description
技术领域
本发明涉及生物技术领域,特别涉及皮肤病理切片的制作方法。
背景技术
皮肤组织弹性大,结构复杂,层次多,不同种属来源皮肤各层组织之间连接松紧水平及各层组织致密程度不同,皮肤组织大致分为表皮、真皮、皮下组织、皮肤附属器等复杂结构,这些组织致密程度不尽相同,角质层相对更致密些,采用常规取材及固定方法处理时皮肤组织容易出现蜷曲、变形,不仅无法还原活体组织的原有形态,切片时也常会遇到只能获取表皮/真皮/皮下组织斜切面而非皮肤全层垂直切面的问题,不仅会造成实验标本的浪费,还会使表皮、真皮或病变组织测量数据出现较大误差。因此,相比其它器官组织的病理检查,皮肤组织病理检测对固定、包埋的要求更高,操作难度更大。
发明内容
为了解决上述问题,本发明提供了一种皮肤病理切片的制作方法,将皮肤组织的取材、固定和包埋方法规范化,解决了病理标本与活体组织形态一致性差、病理图片信息不完整、标本废用的问题,还能在不显著增加操作难度的前提下,为皮肤组织免疫组织化学、免疫荧光染色提供自身对照。
本发明的技术方案如下:
一种皮肤病理切片的制作方法,包括如下步骤:
(1)活体取材:将实验动物吸入适当浓度异氟烷或戊巴比妥腹腔注射诱导深度麻醉成功后,以充分暴露术野、便于操作为目的摆放体俯卧位,常规消毒铺单,剔除实验动物目标区域被毛,剪取目标区域皮肤至对应肌层,得到离体组织,该离体组织包括皮肤、对应皮下组织及深部肌肉;或者离体取材,将死亡的人体或者动物体,以充分暴露术野、便于操作为目的摆放体俯卧位,常规消毒铺单,剔除实验动物目标区域被毛,剪取目标区域皮肤至对应肌层,得到离体组织,该离体组织包括皮肤、对应皮下组织及深部肌肉;
(2)离体组织固定:将离体组织用手术刀将皮肤修为长条形组织块,并将其按原始大小和形状贴附于硬纸片上,将离体组织完全浸泡于10%中性甲醛溶液24小时;
(3)离体组织脱水:将固定好的离体组织于75%酒精1小时,80%酒精1小时,95%酒精I、II各2小时,无水酒精I、II各2小时,二甲苯透明I、II各1小时进行脱水处理;
(4)浸蜡、包埋:将脱水的离体组织置入在60-65℃温箱内已融化的石蜡液I、II缸内各浸1.0-1.5小时,然后进行包埋得到蜡块,包埋时保持组织标本表皮面垂直于包埋底板,且组织标本上下切面与包埋板平行;
(5)切片:在切片机上夹持蜡块时皮肤表皮面朝下,组织面要与切片刀口平行;切片厚度2-5μm,裱片温度以40-45℃为宜,温度过高蜡带易产生气泡和使组织散裂,温度过低蜡带不易伸展,裱片时要求表皮面朝上,易于观察;
(6)染色:根据观察目的按照常规染色方法进行染色。
步骤(4)中,所述浸蜡及包埋用蜡应为同等密度、同等熔点的石蜡,以保证蜡块与组织硬度一致;
步骤(4)中,所述组织标本紧贴包埋底板;
步骤(5)中,所述切片刀要求锋利无缺口,用力要求平和均一。
在取材时,只有取到深部肌肉层,才能确保其浅层皮肤、皮肤创面床组织结构的完整性,否则,当观察对象为全层皮肤创面时,术后早期取材时如不取深至肌肉层,往往会只单独取到一片带创面形状空洞的皮肤,失去最重要的创面床组织;
在离体皮肤组织固定时,由于皮肤富含弹力纤维,松弛,弹性及移动性大,如取材后直接丢入固定液浸泡,组织将在浸泡过程中出现皱缩、卷曲甚至呈球状的情况。这样的标本在后期包埋时难以恢复原状,切片时难以获取具有原本对应关系的皮肤及皮下组织切面,对实验结果观测有极大影响。而将标本贴于硬纸片上再浸入固定液时,标本保持原有形貌,皮肤、皮肤创面,皮下结缔组织、深部肌肉层全部保持原有状态,对实验结果的准确性极为重要,尤其是深度烧伤实验时,还可以观察到烧伤引起的深部肌肉水肿。在后期免疫组织化学染色时,肌肉还可以作为判断背景着色程度是否符合要求的一个指标。
本发明的有益效果是:可以维持皮肤组织内血管的正常形态结构;取材取至肌肉层,可确保标本组织(包括皮肤、皮肤创面/瘢痕、皮下组织及对应深层肌肉)结构的完整性,便于后期观察,用手术刀对组织块进行锐性处理,可避免出现剪刀修剪时因器械挤压而出现的组织结构及各层组织之间相对关系破坏的问题,最大程度的保留组织原貌。修好的组织首先固定于硬纸片上,可最大限度地保持组织原有形貌,避免出现皱缩、卷曲的情况。这些都为皮肤组织病理结果的观察及检测奠定了良好的基础。本发明所涉及的方法可以广泛用于皮肤、皮肤疾病的组织病理学与疾病机制研究,在组织工程皮肤及真皮替代物的临床前检验中也具有重要的实用价值。
附图说明
图1为标记大鼠背部皮肤取材范围图;
图2为离体组织修剪后的状态,A:离体组织的正视图;B:离体组织的剖面图;
图3为修整后的组织浸泡于组织固定液的状态图;
图4为皮肤组织浸蜡、包埋状态图;
图5为低倍镜下取材组织的HE染色结果图。
具体实施方式
通过实施例的描述,对发明的具体实施方式作进一步详细的说明,目的是帮助本领域的技术人员对本发明的构思、技术方案有更完整、准确和深入的理解,并有助于其实施。
实施例1
一种皮肤病理切片的制作方法,包括如下步骤:
(1)大鼠皮肤组织取材:将死亡的大鼠,在实验台上铺加热毯及白纸,将大鼠摆放为俯卧位,取一张蓝色手术单,在中间剪一正方形开口用于暴露手术视野,将开口对准大鼠背部铺于大鼠上方,手术单开口中轴线与大鼠背部中线保持一致;用剃毛机剃除大鼠背部被毛,并使用脱毛膏将取材视野区完全脱毛,将透明膜片放于目标区域上方用油性笔描出并记录目标区域大小形状,见图1;另取一小纸条记录实验时间及实验对象,用眼科剪剪取大鼠背部目标区域皮肤,深至肌肉层次;取下组织后由浅至深可见表皮、真皮及皮下组织;
(2)离体组织的固定:将离体组织用手术刀将皮肤修为长条形组织块,见图2,并将其自然附于硬纸片上,将皮肤病理切片完全浸泡于10%中性甲醛溶液24小时,见图3;
从图2可以看出皮肤创面在离体之后仍保持原有状态,皮肤、皮下及深部肌肉组织保持原有状态,从图3可以看出手术刀修整后的组织浸泡于组织固定液,仍保持良好形态,无明显蜷曲;
(3)离体组织脱水:将固定好的离体组织于75%酒精1小时,80%酒精1小时,95%酒精I、II各2小时,无水酒精I、II各2小时,二甲苯透明I、II各1小时进行脱水;
(4)浸蜡、包埋:将脱水后的离体组织置入在65℃温箱内已融化的石蜡液I、II缸内各浸1.0小时,然后进行包埋得到蜡块,见图4,浸蜡用蜡与包埋用蜡应为同等密度、同等熔点的切片石蜡,以保证蜡块与组织硬度一致;包埋时组织标本表皮与包埋板呈垂直方向,且组织标本上下切面与包埋板平整摆放,组织标本紧贴包埋底板;
从图4可以看出蜡块中取材组织较平整,保持良好形态;
(5)切片:在切片机上夹持蜡块时皮肤表皮面朝下,组织面要与切片刀口平行;切片刀为手术刀,切片厚度3.5μm,裱片温度以45℃为宜,裱片时要求表皮面朝上,易于观察;
(6)染色:采用苏木精伊红染色法进行染色,染色结果见图5。
从图5可看出皮肤、皮下及深部肌肉组织形态保存良好,各层组织对应关系未被取材、固定及包埋操作破坏。
上述实施例中,采用手术刀对离体组织进行修剪,保留完整创面,肉芽可见全层皮肤及皮下组织,皮肤组织浸泡于组织固定液中,保持良好形态,无明显蜷曲,蜡块中皮肤组织较平整,保持良好形态,最终的皮肤病理切片皮肤结构完整。
对于活体取材的方法就是将实施例1步骤1中的死亡大鼠替换为SD大鼠,将适当浓度异氟烷灌入小动物麻醉机中,打开氧气管及小动物麻醉机,待大鼠麻醉,在实验台上铺加热毯及白纸,将大鼠摆放为俯卧位,大鼠持续麻醉,取一张蓝色手术单,在中间剪一正方形开口用于暴露手术视野,将开口对准大鼠背部铺于大鼠上方,手术单开口中轴线与大鼠背部中线保持一致;用剃毛机剃除大鼠背部被毛,并使用脱毛膏将取材视野区完全脱毛,将透明膜片放于目标区域上方用油性笔描出并记录像图1一样的大小形状,另取一小纸条记录实验时间及实验对象,用眼科剪剪取大鼠背部目标区域皮肤,深至肌肉层次;取下组织后由浅至深可见表皮、真皮及皮下组织,步骤2-6与实施例1一致,最后得到的切片与实施例1一致,皮肤、皮下及深部肌肉组织形态保存良好,各层组织对应关系未被取材、固定及包埋操作破坏。
本发明的实施例不仅限于大鼠,还可以猪、兔、猴、人等作为取材对象,可根据实际需求选择取材对象,本发明旨在公开一种皮肤病理切片的制作方法,具有通用性。
Claims (6)
1.一种皮肤病理切片的制作方法,其特征在于,包括如下步骤:
(1)活体取材:将实验动物深度麻醉成功后,以充分暴露术野、便于操作为目的摆放体位,常规消毒铺单,剃除实验动物目标区域被毛,剪取目标区域皮肤至对应肌层得到离体组织;或者离体取材,将死亡的人体或者动物体,以充分暴露术野、便于操作为目的摆放体俯卧位,常规消毒铺单,剔除实验动物目标区域被毛,剪取目标区域皮肤至对应肌层,得到离体组织,该离体组织包括皮肤、对应皮下组织及深部肌肉;
(2)离体组织固定:将离体组织用手术刀修为长条形组织块,并将其按原始大小及形状贴附于硬纸片上,完全浸泡于10%中性甲醛溶液24小时;
(3)离体组织脱水:将固定好的离体组织于75%酒精1小时,80%酒精1小时,95%酒精I、II各2小时,无水酒精I、II各2小时,二甲苯透明I、II各1小时进行脱水处理;
(4)浸蜡、包埋:将脱水的离体组织置入在60-65℃温箱内已融化的石蜡液I、II缸内各浸泡1.0-1.5小时,然后进行包埋得到蜡块;
(5)切片:在切片机上夹持蜡块时皮肤表皮面朝下,组织切面与切片刀口平行;切片厚度2-5μm,裱片温度以40-45℃为宜,裱片时表皮面朝上;
(6)染色:根据观察目的按照常规染色方法进行染色。
2.如权利要求1所述的皮肤病理切片的制作方法,所述步骤(1)中的离体组织包括皮肤、对应皮下组织及深部肌肉。
3.如权利要求1所述的皮肤病理切片的制作方法,所述步骤(4)中的浸蜡及包埋用蜡应为同等密度、同等熔点的石蜡,以保证蜡块与组织硬度一致。
4.如权利要求1所述的皮肤病理切片的制作方法,所述步骤(4)中包埋时保持组织标本表皮面垂直于包埋底板,且组织标本上下切面与包埋板平行。
5.如权利要求1所述的皮肤病理切片的制作方法,所述步骤(4)中的组织标本紧贴包埋底板。
6.如权利要求1所述的皮肤病理切片的制作方法,所述步骤(5)中的切片刀要求锋利无缺口。
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