I have been having a problem with what appears to be rods and stippling on my slides after in situ hybridization. I use NBT/BCIP as my substrate and have counterstained with methyl green or bismarck brown y and it makes no difference. I have also tried incubating my slides in the substrate at RT or at 37C and it also makes no difference. In case it's of interest my antibody is added at a dilution of 1:200 and my substrate solution consists of 15.712 ml Buffer 2 + 72ul NBT + 56ul BCIP+ 160ul 24mg/ml tetramisole-HCl. Has anyone else had this problem?Attached are two pictures that illustrate what I'm explaining.
I use Genpoint(R) from Dako with DAB as substrate counterstain with hematoxylin.
Hi Heather,
Do you use Polyvinylpyrrolidone or some similar polymer for your staining reaction? I get a similar effect if there is some of the PVP left on the slide. Best Simone
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