----李紅麗第三軍醫大學組織學與胚胎學教研室Email:

lihlimm@免疫組化實驗中的注意事項一、Specimenpreparation

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

ParaffinSectionsFixation

Objective:Topreservecellsandtissuesinalife-likemanner保持形態結構，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、固定組織時的注意事項應力求保持組織新鮮，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3、Karnovsky液(PH7.3)多聚甲醛30g25%戊二醛80ml0.1mol/LPB至1000ml電鏡免疫細胞化學，可中長時間固定，較好地保存抗原和細微結構。丙酮或乙醇：培養細胞涂片標本新鮮

2周內；抗原修復:微波，高壓，檸檬酸緩沖液Cryosectioning

方法：冰凍時，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組織結構保存良好，能切連續薄片，組織結構清晰，抗原定位準確。易于保存標本。Disadvantages脫水、透明等過程最好在4℃，組織塊應較小（厚度小于0.2cm），浸蠟包埋等應保持在60℃以下。1.Fixsmallblocks(10x10x3mm)oftissue(usuallyinformaldehyde)forupto24hrs.2.Processroutinelytoparaffin.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）.對抗原部位屏蔽的程度與固定時間、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。1.ProteolyticEnzymeDigestionThecrosslinkscanbepartiallydisruptedbyproteolyticenzymes(trypsin胰蛋白酶).Trypsinizationtimeisextremelyimportantandisproportionaltothespecimenfixationtime.胰蛋白酶在37℃和pH7.8時活性最佳.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,assomeantigensmaybealteredordestroyedbytrypsinization.immunostainingmaybeimpairedorcompletelyremovedfollowingtrypsinisation.Proteolyticdigestionhaslargelybeenreplacedbyheatmediatedantigenretrievalmethods.ProtocolofTrypsinretrievalplacesectionsinprewarmed(37oC)distilledwater.prepare0.1%trypsinin0.1%calciumchloride:pre-warmeddistilledwater(400ml)

Trypsin(100mg)5%calciumchloride(8ml)AdjusttopH7.6with1%sodiumhydroxide.3.incubatesectionsfortherequiredtime:resinsections:6minutes.paraffinsections(trypsinonly):3minutes.paraffinsections(trypsin+MW):1minute.4.washsectionsincoldwatertopreventfurtherdigestion.

2.HeatMediatedAntigenRetrieval原理:Onetheoryisthatheavymetalsalts

forminginsolublecomplexeswithpolypeptidesandthatproteinprecipitatingfixativesfrequentlydisplaybetterpreservationofantigensthandocross-linkingaldehydefixatives.Anothertheoryisthatduringformalinfixationinter-andintra-molecularcrosslinkagesareformedbymethylenebridgesandweakSchiffbases.ItispostulatedthatheatmediatedantigenretrievalremovestheweakerSchiffbasesbutdoesnotaffectthemethylenebridgessothattheresultingproteinconformationisintermediatebetweenfixedandunfixed.ProtocolofMicrowaveRetrieval1.placesectionsin400mlof10mMcitratebufferpH6.0.2.(i)paraffinsectionsundergoingtrypsinizationandmicrowavepre-treatment:microwaveonhighpower(800w)for9minutes,washinwater.(ii)paraffinsectionsusingmicrowaveirradiationonly:microwaveonhighpowerfor12minutes,washinwater.(iii)resinsections(withorwithoutpriortrypsinization):microwaveonhighpower(