固定液,冷冻切片, 急性炎性反应,免疫组织化学染色," /> 固定液,冷冻切片, 急性炎性反应,免疫组织化学染色,"/> Fixative, Frozen section, Acute inflammation,Immunohistochemistry,"/> <span style="line-height:2;font-size:14px;">6 种固定液对小鼠皮肤急性炎性反应冷冻</span><span style="line-height:2;font-size:14px;">组织切片免疫组织化学染色效果的比较</span>
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发育医学电子杂志  2020, Vol. 8 Issue (1): 29-33    DOI: 10.3969/j.issn.2095-5340.2020.01.006
  结构畸形   论著 |
6 种固定液对小鼠皮肤急性炎性反应冷冻组织切片免疫组织化学染色效果的比较
宋岩彪 杜娟 张创 曹学会  靳晓次 刘雪来
1. 河北医科大学第二医院中心实验室,石家庄 050000;2. 吉林省人民医院 内分泌科,长春 130021;3. 河北医科大学第二医院 小儿外科,石家庄 050000;4. 河北医科大学第二医院 动物实验中心,石家庄 050000;5. 首都儿科研究所附属儿童医院 外科,北京 100020
Comparison of six differential fixatives on immunohistochemistry staining of frozen tissue sections in mouse skin with acute inflammatory responses
SONG Yan-biao, DU Juan , ZHANG Chuang, et al
1.Department of Central Laboratory, The Second Hospital of Hebei Medical University, Hebei, Shijiazhuang 050000,China; 2.Department of Endocrinology, Jilin Province People’s Hospital, Jilin, Changchun130021, China;3.Department of Pediatric Surgery, The Second Hospital of Hebei Medical University, Hebei, Shijiazhuang050000, China; 4.Department of Animal Experimental, The Second Hospital of Hebei Medical University,Hebei, Shijiazhuang 050000, China; 5.Department of Surgery, Children's Hospital Capital Institute of Pediatric, Beijing 100020, China)
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摘要 【摘要】 目的  探讨不同固定液对小鼠冷冻切片免疫组织化学染色的影响,筛选出相对适合的固定液。方法 选取Balb/C 小鼠2 只,制作皮肤炎性反应模型。小鼠麻醉后剃除背部皮肤表面的毛发,局部暴露于70 ℃热水中35 s,形成1 处1.5 cm×1.5 cm 的深Ⅱ°皮肤烫伤,第7 天取材,部位为以烫伤为中心的大小2.5 cm×2.5 cm 全皮层组织,包埋后冷冻连续切片。取1 组切片,即刻进行免疫组织化学染色,EAF(85% 丙酮+10% 甲醛+5% 醋酸)、AAF(85% 无水乙醇+10% 甲醛+5% 醋酸)、甲醛钙(100%
甲醛+1% 氯化钙)、甲醛(10%)、丙酮(100%)、乙醇(95%)6 种固定液分别染色4 张,2 张切片不经固定液处理;取另一组切片,先采用丙酮、乙醇分别固定4 张,另有2 张切片不经固定液处理,再在-20 ℃保存14 d 后取出进行免疫组织化学染色,检测表达于巨噬细胞胞浆的F4/80 和表达于细胞膜表面的肿瘤坏死因子-α(TNF-α)受体。 结果  小鼠皮肤炎性反应模型造模成功,HE 染色结果提示炎性反应。即刻染色的切片中,F4/80 表达结果显示,AAF、EAF、甲醛、甲醛钙固定切片为阴性,丙酮、乙醇固定和未固定切片为阳性;TNF-α 表达结果显示,AAF、EAF 固定切片为阴性,甲醛、甲醛钙、丙酮、乙醇固定和未固定切片为阳性。低温保存2 周后,丙酮、乙醇固定切片的TNF-α 阳性表达低于即刻染色组,但F4/80 表达未见明显差异。 结论 不同固定液对冷冻切片的免疫组织化学染色结果有影响。丙酮和乙醇固定液更适用于炎性组织冷冻切片的固定,是相对理想的固定液。
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关键词:  font-size:14px  固定液')" href="#">">固定液  冷冻切片  急性炎性反应  免疫组织化学染色    
Abstract: 【Abstract】 Objective To explore the effects of different fixatives on immunohistochemistry staining in frozen tissue sections of mouse, and screening more reliable fixation. Methods Two Balb/C mice were used to create the animal model of skin with acute inflammation. The hair on dorsal skin of mice were eliminated and part of those were exposed to hot water at 70℃ for total of 35 seconds after anesthesia,creating deep skin scald with square shape (1.5 cm×1.5 cm). Whole cortex tissue with square shape(2.5 cm×2.5 cm) around scalded skin was picked up on the seventh day , and then embedded into frozen
serial sectioning. One group of slices were taken for immunohistochemistry staining immediately. Four sections were exposed by each of the six different fixatives[EAF (85% acetone + 10% formaldehyde +5%ethylic acid), AAF (85% absolute alcohol + 10% formaldehyde + 5% ethylic acid), calcium formaldehyde(100% formaldehyde + 1% calcium chloride), formaldehyde (10%), acetone (100%) and ethanol (95%)]and the other two slices were treated without fixation. In another group, four sections were treated by acetone and four by ethanol, meanwhile two sections were treated without fixation. The later group was
stored at ﹣ 20 ℃ for 14 days, then was strained by immunohistochemistry. F4/80 (expressed in cytoplasm)and TNF-α (expressed in cytomembrane) monoclonal antibodies were used for immunohistochemistry staining. Results Mice model of skin with acute inflammation were created successfully and confirmed inflammation by HE staining. F4/80 staining was negative in AAF/EAF/formaldehyde/calcium formaldehyde of fixative sections in immediately stained sections, unfixed sections and fixation of acetone and ethanol were positive. TNF-α staining was negative in AAF/EAF fixative sections, the sections of unfixed and fixative
in formaldehyde, calcium formaldehyde, acetone and ethanol were positive. TNF-α positive expression in fixative sections of acetone and ethanol after keeping two weeks were lower than those in immediately stained group, but no significance were found in F4/80 staining. Conclusions The different fixatives will have influence on immunohistochemistry staining of frozen sections. The acetone and alcohol are the relative ideal fixative and more applicable to fixate frozen tissue sections with inflammatory responses.
Key words:  font-size:14px    Fixative')" href="#">">Fixative    Frozen section    Acute inflammation    Immunohistochemistry
收稿日期:  2019-01-30                出版日期:  2020-01-30      发布日期:  2020-01-22      期的出版日期:  2020-01-30
基金资助: 河北省医学适用技术跟踪项目(G201720)
通讯作者:  刘雪来    E-mail:  liuxuelai_steven@163.com
引用本文:    
宋岩彪 杜娟 张创 曹学会  靳晓次 刘雪来. 6 种固定液对小鼠皮肤急性炎性反应冷冻组织切片免疫组织化学染色效果的比较[J]. 发育医学电子杂志, 2020, 8(1): 29-33.
SONG Yan-biao, DU Juan , ZHANG Chuang, et al. Comparison of six differential fixatives on immunohistochemistry staining of frozen tissue sections in mouse skin with acute inflammatory responses. Journal of Developmental Medicine(Electronic Version), 2020, 8(1): 29-33.
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