標(biāo)本取材與固定

----李紅麗第三軍醫(yī)大學(xué)組織學(xué)與胚胎學(xué)教研室Email:

lihlimm@免疫組化實(shí)驗(yàn)中的注意事項(xiàng)一、Specimenpreparation

取材新鮮，固定及時(shí)，形態(tài)保存完好，抗原物質(zhì)的抗原性不丟失、不擴(kuò)散和被破壞Fixation：灌注固定和浸泡固定SampleTypeCryostat(frozen)sections

ParaffinSectionsFixation

Objective:Topreservecellsandtissuesinalife-likemanner保持形態(tài)結(jié)構(gòu)，防止自溶afalselocalization,anegativeresult

Methods:byperfusionand/orimmersion

Theeasiestmethodischemicalfixation:aldehydes

Thetwomostpopularaldehydesbeingformaldehyde甲醛

（upto8%15-60min

）

andglutaraldehyde戊二醛（upto1%for15-60min

）Characteristicsofformaldehyde1.lowconcentrations(<4%)formaldehydecross-linkingispartlyreversible.Itisimportanttoavoidextensivewashing.2.thecross-linkingreactionsofformaldehydeoccurmuchslower.Itisbesttoleavethecellsortissuestobefixedforalongertime.3.Formaldehydeexistsinsolutionasmonomersandpolymersandthepolymersaremoreactiveatcross-linking.Thepolymersarepresentinhighernumbersinmoreconcentratedsolutionsandwhencooledto4℃offormaldehyde.1、固定組織時(shí)的注意事項(xiàng)應(yīng)力求保持組織新鮮，勿使其干燥，盡快固定處理組織塊不宜過大過厚，厚度必須在0.3cm內(nèi)足夠量的固定液，其體積大于組織20倍固定后充分水洗，以減少固定液造成的人為假象常用的固定劑1、改良Bouin液飽和苦味酸750ml40%甲醛250ml組織的穿透性較強(qiáng)，不適合長(zhǎng)期保存2、Zamboni液多聚甲醛20/30g飽和苦味酸150mlK-SPB至1000ml細(xì)胞的抗原及超微結(jié)構(gòu)均保存較好3、Karnovsky液(PH7.3)多聚甲醛30g25%戊二醛80ml0.1mol/LPB至1000ml電鏡免疫細(xì)胞化學(xué)，可中長(zhǎng)時(shí)間固定，較好地保存抗原和細(xì)微結(jié)構(gòu)。丙酮或乙醇：培養(yǎng)細(xì)胞涂片標(biāo)本新鮮

2周內(nèi)；抗原修復(fù):微波，高壓，檸檬酸緩沖液2、SampleTypeThetwocommonoptionsarecryostat(frozen)sectionsandparaffinsections.cryostat：Advantagesexcellentantigenpreservationandanyfixativecanbeused.Disadvantageslessmorphologicaldetailandresolution（分辨率）Cryosectioning

方法：冰凍時(shí)，組織中水份易形成冰晶，影響抗原定位。1.液氮速凍法：2.蔗糖高滲法（20～30%）

1.6-2.3Mol/L

15-60min

sucroseasacryo-protectantafterfixationbutpriortofreezing.vitrified（玻璃狀）(i.e.noicecrystalsformed)state

ProtocolofCryostat(frozen)sections1.Snap-freezesmalltissueblocks(5x5x3mm)inliquidnitrogen.2.Transfertocryostatandcutthinsections.3.Collectspecimensoncleanpoly-L-lysine-coatedglassslidesanddryatroomtemperatureovernight(ifyouwanttostainthesamedayletair-dryfor1-2hr).4.

Fixinacetoneat4℃orabsoluteethanolfor15min.5.Air-dry.6.Proceedwithimmunostaining.ProtocolofParaffinSectionsAdvantages組織結(jié)構(gòu)保存良好，能切連續(xù)薄片，組織結(jié)構(gòu)清晰，抗原定位準(zhǔn)確。易于保存標(biāo)本。Disadvantages脫水、透明等過程最好在4℃，組織塊應(yīng)較?。ê穸刃∮?.2cm），浸蠟包埋等應(yīng)保持在60℃以下。1.Fixsmallblocks(10x10x3mm)oftissue(usuallyinformaldehyde)forupto24hrs.2.Processroutinelytoparaffin.二、增強(qiáng)特異性染色方法孵育溫度/時(shí)間：大多37℃/30-90min，4℃/18h，roomtemperature/1-6h注意保持濕潤(rùn)，防止干燥抗原修復(fù)多層染色法顯色增敏劑：如在過氧化物酶底物中加入氯化鎳，或硫酸鎳胺等可提高顯色敏感度4倍AntigenRetrievalTechniquesIntention:Tofacilitatetheimmunologicalreactionofantibodieswithantigens(increasereactivityofthemajorityofantigens).以formalin固定為例Principles:duetotheformationofmethylene(亞甲基)bridgesbetweenreactivesitesontissueproteins,inter-andintra-molecularcrosslinkswithcertainstructuralproteinswhichareresponsibleforthemaskingoftissueantigens.Thesereactivesitesincludeamines（胺）,amide（酰胺）,thiols(硫醇),alcoholichydroxyl（羥基）groupsandcyclicaromaticrings（芳香基環(huán)）.對(duì)抗原部位屏蔽的程度與固定時(shí)間、溫度、固定劑濃度及抗原附近其它易形成鉸鏈的蛋白的存在有關(guān)。Thethreemostcommonantigenretrievaltechniquesare

1.ProteolyticEnzymeDigestion2.HeatMediatedAntigenRetrieval)

MicrowaveIrradiationAutoclavingorPressureCooking1.ProteolyticEnzymeDigestionThecrosslinkscanbepartiallydisruptedbyproteolyticenzymes(trypsin胰蛋白酶).Trypsinizationtimeisextremelyimportantandisproportionaltothespecimenfixationtime.胰蛋白酶在37℃和pH7.8時(shí)活性最佳.Thereactionrateisimprovedbytheadditionoftheco-enzymecalciumchloride(0.1%).Trypsinonlyremainsactiveforabout30minutes,

Enzymesusedinclude

pronase(鏈霉蛋白酶)(0.05%(w/v)inPBS),

trypsin胰蛋白酶(0.05%(v/v)inPBSwith0.1%CaCl2)

pepsin(胃蛋白酶)(0.05%(v/v)in2NHCl).Theconditionsofconcentration,timeandtemperaturemustbecontrolled.

Disadvantagescreating"false"antigenicsites,assomeantig