SPAM-LOW: [Histonet] RE: Granzyme b and F4/80

[Patsy Ruegg]

Jennifer,

Your NS background could be coming from endogenous biotin.  I use the rat
anti ms F4/80 on ffpe mouse tissue all the time and it is very clean but I
use a labeled polymer detection system rather than AB.  I use rat on mouse
hrp or ap labeled polymer detection from BioCare, or I use my own link
secondary that is rab anti rat and then follow with hrp rabbit labeled
polymer (PolyVision from Leica).  I do use a protein block before the
primary but no serum block.

Best regards,

Patsy 


Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 215
Aurora, CO 80010
P-720-859-4060
F-720-859-4110
email pruegg <@t> ihctech.net
website www.ihctech.net
IHC Resource Group www.ihcrg.org 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of PALMER Jason
(SVHM)
Sent: Wednesday, October 21, 2009 6:12 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: SPAM-LOW: [Histonet] RE: Granzyme b and F4/80


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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