I am doing an IHC protocol on a testes FFPE tissue slide. For primary Ab I am using Anti-Geminin antibody [EPR14637] from Abcam. For secondary conjugate I am using HRP. For chromogen I am using DAB. I am not co-staining the slides because I just want to look at geminin expression. The geminin ab is new and works well, same with HRP. I am following the general IHC protocol but at the end of the dehydration step and after mounting the slides, I am not seeing any staining develop. It's so weird, because I can see the reaction happening and the tissue getting darker with stain after washing and dehydrating them, the staining just vanishes.
Can anyone please help me with this?
Dear Sharanya,
How do you perform the dehydration step?
I usually don't perform it in my DAB stainings: after the staining I wash my sections with PBS 3 times x 5', mount them on the slides and then I wait for the sections to be dry. At that point I just coverslip them with NeoMount.
Note well: my sections are formalin fixed.
Dear Sharanya,
It's actually unusual that the staining vanishes during dehydration. DAB is insoluble to water, alcohol and xylene. May be the colouring you might have noticed is not the specific. It would suggest you to first do a western blotting so as to confirm the expression of the protein of your interest. In case you get the band then you shall proceed with a trouble shooting.
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