I am doing a double-staining of TnI and cleaved-caspase-3 in paraffin-embedded mouse heart sections. In negative where there's no primary, I see a great deal of background of the secondary for the caspase-3 (alexa 488). What can I do to get rid of this high background 2ndary staining? Should I cook more or use different antigen retrieval solution?
primaries: m-TnI and Rb-m Caspase-3
secondaries: G-m Alexa 546 and G-Rb Alexa 488
block - Rodent Block M (biocare)
Antigen retrieval solutions tried: citric acid buffer pH-6, high pH Vector unmasking solution and Rodent Decloaker.
Hi
How long you are incubating the sections with blocking solution?
Which solution are you using for wash after 2nd ab incubation? How long is the washing time?
Thnx
Viz
Thanks for your reply, Vijayendra!
The sections are blocked for 30min with Rodent Block M. I'm increasing it up to 1hr today and see what happens.
I use PBS wash after 2ndary for 4 times, 5' each (total of 20')
-Duke
Hi Duke
I would recommend nearly 1h blocking. In addition, diluting primary as well as secondary antibodies in a 50-50 mixture of blocking solution and PBST (0.1% triton-x-100) helps.
For washing also (after primary as well as secondary), I prefer PBST solution.
Hope it helps!
Viz
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