gelatin

Immunofluorecence

IFStainingwithParafilmSectionSlide

Procedure:

ffinize/hydrateusingthefollowingriesofwashes:

Xylenes4X15minRT

100%EtOH2X30min

99%EtOH5min

95%EtOH5min

90%EtOH5min

80%EtOH5min

70%EtOH5min

50%EtOH5min

dH2O5min

PBS5min

2Treatedwithblockingbuffer(BSA5%,NGS1%,NSS1%,Tritonx-1000.1%fish

gelatin0.2%inPBS)for60minat32C.(beforethat,drawacycleoneachslidewith

specialpen)

thPBS10min3X

rimaryAntibodyin1:200inblockingbuffer(BSA1%,Tritonx-1000.1%

inPBS)RTovernight.

thPBS10min3X

oatanti-rabbit546andavidinbindAlex488for2hrsatRT.1:500

thPBS10min4X,onshaker

ngtheslidewithMowiolmoutingsolution

darkboxat4ºC.

Mountingsolution:

MOWIOL5.0gCalbiochem;475904

+PBS20ml(含NaN3)

StirOvernightat4C

+glycerol10ml

Stirovernightat4C

Spindown,15000rpm,30minat4C

Aliquots,1.0mleach,storeat-20C

BlockingSolution:

BSA(Sigma)1g(2%)

FCS1ml(2%)

Gelatin(Amersham)100ul(0.2%)RPN416

PBSTo50ml

Aliquots,10mleach

Blockbuffer:

BSA(sigma)0.5g

NGS100ul

NSS100ul

Tritonx-10010ul

PBS9.8ml

Stiruntildissolved

Skimmilk0.5g

Stiruntildissolved

Gelatin20ul

Antibody:

IM409-434