[Histonet] RE: Granzyme b and F4/80

[PALMER Jason (SVHM)]

For Serotec F4/80, we use 10% rabbit serum as block and 5% of the same as antibody diluent  -our secondary is a biotinylated rabbit anti rat.  We also retrieve with Dako proteinase K rather than citrate, with simple HRP-streptavidin then DAB, and it all works very nicely, no background issues.

Hope this helps,

Jason

Jason Palmer
Histology Laboratory Coordinator
O'Brien Institute
42 Fitzroy St, Fitzroy Victoria 3065
Australia
tel +61 3 9288 4045
fax +61 3 9416 0926
email: jason.palmer <@t> svhm.org.au


Hi All,

 I'm currently trying to work up Granzyme B and F4/80 on FFPE mouse tissue.  I seemed to get a bit of non-specific staining for both and was wondering if there is any way I can eliminate some of this.  For Granzyme B I am using a rabbit polyclonal from Abcam.  My protocol goes like this: H202 block, citrate buffer HIER, Powerblock (casein-based, non-serum protein block), primary antibody, secondary antibody (rabbit-on rodent HRP polymer, from biocare), DAB, counterstain.  My F4/80 is a rat anti-mouse primary from Serotec and the protocol is as follows: H202 block, citrate buffer HIER, Powerblock, avidin/biotin blocks, primary antibody, secondary antibody (Goat anti-rat, biotinylated, mouse-absorbed, from Biocare), HRP Label, DAB, counterstain.  Is it true that if I switched to a serum block from the species in which the secondary was raised, instead of my non-serum protein block, it may help?  Thank you in advance!

Jennifer

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