I stained a bone tissue with TRAP and counterstained with Mayers hematoxylin for 5 seconds. After rinse in running tap water, the result was just fine. However after I dried the section for some minutes before mounting the slides, the section become so black and I could not see the osteoclast clearly. I have tried to rinse the slides with 0.25% hydrochloric acid for 5 seconds then keep the slides in running water for 3 minutes, and the colour become acceptable again. Unfortunately, the slides turned black again after a few minutes. Is there any way retrieving the section back to normal?
Why are you drying this, and how are you drying? Are you using a substituted Naphthol substrate for the TRAP staining? What is the colour of your final reaction product? If you use Hexazotised pararosanilin as the diazonium salt for this demonstration, then you can dehydrate through alcohol and clear through xylene to mount in a resinous medium. I have a feeling that your final reaction product may be alcohol/solvent labile, hence the need to dry the sections properly. If the final reaction product is labile to alcohol/solvent, then you may find your choice of mountant is limited The problem is that if you try to dry bone sections, it is likely that you will get microscopic bubbles trapped in the section which are very difficult to remove in bone. This can give the appearance that the section "turns black" - In fact it is full of lots of very small bubbles which will appear black and distort the image you see under the microscope. This is supported by your observation that "the colour becomes acceptable again, but then the section turns "black "when you try to dry it again, yes?
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