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

[Megan French]

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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