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1、北京大學(xué)學(xué)報(醫(yī)學(xué)版)JOURNALOFPEKINGUNIVERSITY(HEALTHSCIENCES)Vo1.43No.6Dec.2011·技術(shù)方法·石蠟切片免疫熒光法在腎病穿刺組織中的應(yīng)用張燕,陳劍,劉海靜,陸敏(北京大學(xué)基礎(chǔ)醫(yī)學(xué)院病理學(xué)系,北京100191)[摘要]目的:探討石蠟切片免疫熒光法檢測腎病穿刺組織中免疫球蛋白等指標(biāo)的最佳實(shí)驗(yàn)條件。方法:收集北京大學(xué)基礎(chǔ)醫(yī)學(xué)院病理學(xué)系的腎病穿刺組織標(biāo)本45例,包括IgA腎病20例、膜性腎病15例、膜型乙肝病毒相關(guān)性腎炎10例,將組織標(biāo)本用10%(體積分?jǐn)?shù))中性甲醛溶液固定、石蠟包埋、組織切片,在
2、不同的抗原修復(fù)條件和抗體結(jié)合時間下,分別標(biāo)記IgG、IgA、HBsAg、HBcAg,觀察染色結(jié)果,并與其新鮮組織的免疫熒光染色結(jié)果相比較,評判石蠟切片免疫熒光法的應(yīng)用價值。結(jié)果:熱誘導(dǎo)抗原修復(fù)法修復(fù)后一步法和二步法染色均為陰性;蛋白水解酶消化法中胰蛋白酶優(yōu)于胃蛋白酶,并以胰蛋白酶消化30rain、IgG和IgA37℃溫箱孵育90min、HBsAg和HBcAg二抗37℃溫箱孵育150min染色結(jié)果最佳,與新鮮組織標(biāo)本染色強(qiáng)度接近或略弱一些,陽性表達(dá)的方式也與后者相同,符合率100%。結(jié)論:經(jīng)胰蛋白酶消化的石蠟切片免疫熒光法能夠較好地應(yīng)用于。腎病穿
3、刺組織診斷。[關(guān)鍵詞]熒光抗體技術(shù);石蠟;活組織檢查;腎臟病學(xué)[中圖分類號]R692[文獻(xiàn)標(biāo)志碼]A[文章編號]1671—167X(2011)06-0900-03doi:10.3969/j.issn.1671—167X.2011.06.023UseofparafinsectionsforimmunofluorescencestaininginrenalbiopsyZHANGYan,CHENJian,LIUHai.jing,LUMin(DepartmentofPathology,PekingUniversitySchoolofBasicMedic
4、alSciences,Beijing100191,China)ABSTRACTObjective:Toexplorethebestmethodfordetectingimmunoglobulin,HBsAgandHBcAginformalin—fixedparaffin—embeddedrenalbiopsytissuesbyimmunofluorescenee(IF).Methods:Inthestudy,45casesofrenalbiopsywerecollected.including20casesofimmunoglobulinA(I
5、gA)nephropa—thy.15casesofmembranousnephropathyand10casesofmembranoustypehepatitisBvirusassociatednephritis.IgG,IgA,HBsAgandHBcAgdepositionswereevaluatedinthesecasesbyIFwithdifferentantigenretrievalmethodsanddifferentperiodsofantibodyincubationtime.TheresultswerecomparedwithI
6、Fonfrozentissuesfromthesamesetofcasestoevaluatethemethodology.Results:A11thecaseswerenegativewithheat.inducedepitoperetrievalalone.Immunoglobulin.HBsAgandHBcAgweremorereadilyidentifiableafterdigestionwithtrypsinthanwithpepsin.IgGandIgAwerebestvisualizedafter30minofdigestionw
7、ithtrypsinand90minofincubationwithprimaryantibodyforIgGandIgA.HBsAgandHBcAgwerebestvisualizedafter30minofincubationwithprimaryantibodyand150minofincubationwithsecondaryantibody.Comparingimmunoglobulins.HBsAgandHBcAgonfreshsectionsandpar舶nsections.theirvisualizationswerealmos
8、tidenticalwithbothtechniquesalthoughthesignalonparaffinsectionswasslightlyw