[Histonet] positive staining on my secondary only controls...need a DAB superquench?

[Elizabeth Chlipala]

Megan

Pre absorbing your antibody for a control for IHC is not always going to work there are publications out there that do not recommend it.  I'll try to find the reference I'm talking about.  I pulled this out of one of my presentations

Protocols available on the web - for western blots

http://www.upstate.com/misc/protocol_detail.q.prot.e.peptide-competion.a.name.e.Peptide_Competition

http://store.crpinc.com/prot_peptide_comp.aspx

"The absorption control is less important because if cannot determine whether the protein bound in the tissue is the same protein that used for absorption.  Therefore, the recommended guidelines for immunohistochemistry include negative controls and positive controls but do not include absorption controls."

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308-1592
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
www.premierlab.com

Ship to address:

1567 Skyway Drive, Unit E
Longmont, CO 80504

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Megan French
Sent: Sunday, July 17, 2011 9:45 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] positive staining on my secondary only controls...need a DAB superquench?



Having some trouble with my IHC optimisation. Hopefully it's a simple
fix someone may be able to provide some guidance (Im a junior RA! Hello
histoworld!).

Im using a DAB protocol with swine-anti-rabbit HRP on human colon. I
have successful staining of my primary (yay!), somewhat successful
staining with my antibody preabsorbed with peptide (yet still some
staining)...BUT im seeing positive staining on my secondary only
controls (may explain why im getting a bit of staining on my preabsorped
specimens). I quench with 5% hydrogen peroxide for 5 min on day 1 (prior
to primaries/no primary if secondary control) and on day 2 add 1 DAKO
DAB chromogen tablet to 10ml PBS and 10ul hydrogen peroxide again before
immersing slides for 8 min, then stain with haematoxylin per usual,
dehydrate, clear & mount. I feel like I need a super quencher or
something?! Any tips would be most helpful. (My slides also undergo
antigen retrieval in citrate buffer ph6 for 10min @ 95deg and 20min at
RT)





Miss Megan French BBiomedSc(Deakin); BSc(Hons)(Melbourne)

Research Assistant

Surgical Research; Infection & Immunity Theme



Murdoch Children's Research Institute

The Royal Children's Hospital
Flemington Road Parkville Victoria 3052 Australia
E megan.french <@t> mcri.edu.au





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