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)
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.
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.
2020, Vol. 8 
