AntibodyLabeling

ThinsectionsofBIOLOGicalmaterial,mountedonspecimengrids,canbeeasilylabeledbyfloatingthem,section-sidedown,onsmalldropsofantibody.Thismethodwillworkforsectionsthroughresin-embeddedmaterialaswellasforthin,thawed,cryosections.

Procedure

IfalongpieceofParafilmisstucktoabenchbenchsurfacewithadropofwatertheantibodyincubationsandwashingstepscanbeperformedonthecleansurfaceoftheparafilm.Onlyexposeasmuchoftheparafilmasitisneededanddonotcontaminatethecleanparafilmsurface.Antibodylabelingprotocolsareavailableontheantibodylabelingpage.Asimplelabelingprocedureissummarizedbelow.

1. Blockingstep5min
2. Antibody15min
3. PBSWash6x2min
4. ProteinAGold15min
5. PBSWash6x2min
6. WaterWash5x1min.

Blocking

Any"sticky"proteincanbeusedtocovernon-specificbindingsitesonthethawedsections.Beawarethatthechosenproteinmustnotreactwithanyoftheaffinityreagentstobeused(e.g.rabbitserumcannotbeusedwithproteinAgold;bovineserum(FCS)cannotbeusedasablockingagentifanti-BSAlabellingisbeingstudied).

Weroutinelyuse10%FCSinPBSasablockingagent,butBSA,gelatin,ovalbuminandcoldwaterfishskingelatinhavebeenused.TheblockingagentsusedonWesternblotsshouldalsoworkonthesesectons.

To"quench"freealdehydegroupsthatmaybepresentinthesection0.15%glycinecanbeaddedtotheblockingsolution.

Antibodylabeling: Specimengridsarefloatedwiththesidecontainingthesectionsontheliquid.DropsofPBS,ordilutedantibodies,areplacedonthecleansurfaceofparafilm.Thegridsaretransferredfromdroptodropontheparafilm.Makesurethesectionsdonotdryout.Theparafilmiskeptcovereduntilreadytouse.

AntibodyIncubation

Theantibody,dilutedtoasuitableconcentration,inPBScontainingblockingagent,iscentrifugedfor1mininanEppendorftubebeforeuse.Thiswillremoveanyaggregatesformedduringstorage.

Placesmalldropletsofantibody(3to5microliter)ontheparafilmsurfaceandfloatthegrids,withsectionsdown,onthem.Thegridsaretransferredfromtheblockingsolutionusingfineforceps.Toensurethattheantibodydropletsdonotdryout(andconcentrate)placeawetpieceoffilterpapernearthemandcoveritandthemwithaplasticdish.

PBSWash

Afterantibodyincubationthegridsareremovedfromtheantibody,withforceps,andplacedontolargedropsofPBSontheparafilmsurface.Transferthegridsfromdroptodropeitherwithforcepsoralargewireloop.Takecarenottowetthetop,drysurfaceofthegrid.

ProteinA-goldIncubation

Liketheantibody,theproteinA-goldisdilutedinPBSwithblockingagentbutitisnotcentrifugedpriortouse.SmalldropletsofdilutedproteinA-gold(samevolumesasusedfortheantibodies)areplacedontheparafilmsurface,thegridsareplacedonthedropsandtheyareleftcoveredduringtheincubation.

GohereifyouwanttoseeamethodofmakingcolloidalgoldprobesandcouplingthemtoproteinssuchasproteinA.

PBSWash

Thisisthesameaswhenwashingafterantibodyincubation.Againtakecarenottowetthebackofthegrid.

WaterWash

Althoughonlyashortwash,THISSTEPISVERYIMPORTANT.DONOTFORGETIT!Thisstepremovesallphosphatemoleculespriortoincubationwithuranylacetate.Phosphatespresentinthesectionswillprecipitatetheuranylsalts.

Oncetheantibodyreactionshavetakenplacetheonlythinglefttodoisthefinalcontrastinganddryingstepwhichwillenableustoexaminethesectionsintheelectronmicroscope.