I am performing IHC staining for CD3 and CD8 in mouse melanoma samples using an AP red system. Each sample has two sections per slide one I stain with CD3 and one with CD8. I use spleens for my positive control. Previously my staining protocol has worked very well, but I have recently encountered a perplexing issue. My positive control tissues (spleen) have been working fine. However, the CD8 staining in tumor sections appears to be largely washing out during my first xylene wash. I monitored the chromagen step and I see clear staining in my CD8 sections that disappear when I look after mounting my slides and my first xylene rinse has taken on a slight pink coloration. A serial section with CD3 stains beautifully but the CD8 is apparently lost. Has anyone experienced similar issues? I am going to try an aqueous mounting solution so I can bypass the xylene rinse, but I would still like an explanation if anyone has one. Thank you!
Hello Justin, no wonder because the red dye which you used for your AP detectios is New Fuchsin (c/o Isorubin, Fuchisin NB, New magenta). This dye is soluble in organic solvents like Xylol. I recommand to use a water soluble mounting medium. You can use it after you have finished the staining . Rinse the slides several times in destilled water, air dry them for ashort time and then mount them.
If you prefer dehydration and clearing in Xylol, then you should use BCIP/NBT (5-Brom-4-Chlor-3-Indolylphosphate). It's a blue colour. Best regards
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