immunohistochemistry - (Dec/09/2005 )

I'm trying to stain mouse bladder with anti-myosin and anti-vimentin and the only time I can get signal on muscle, I have some on urothelium too... Can somebody help me with that ? What can I do to optimise my staining ? Thanks,

I can help if you give me details of the protocol. Let's start there.

For vimentin, i'm not doing any antigen retrieval and i'm using dilution 1/50 of the Ab clone RV202 from Abcam. Second antibody is IDlabs IDetect kit. We need to use fresh slide to stains, if not, we get no signal at all.

For myosin, i'm doing heat antigen retrieval (6 min microwave) followed with prot K enzyme digestion (0,01% 10 min RT) and i'm using dilution 1/100 -1/250 of the Ab clone SMMS-1 from Chemicon. Second antibody is also IDlabs IDetect kit.

My real problem is the non-specific staining of the urothelium ... When I go with more dilution, I lost both of signal... in muscle as in urothelium... Does it mean that these antigen are present in urothelium or that my Abs are non-specific ? Or again, my conditions are not right ?

Thanks,
Jolyann