Hi everyone, I am trying to deal with an unreasonable problem with TRAP staining of differentiated RAW264.7 cells (RANKL induced). I was using a protocol for TRAP staining, and it worked perfectly fine before. But now, I am using the exact same protocol and all of a sudden my differentiated cells are not stained purple, everything is some kind of green? What could be the reason for it? Did the reagents degrade? PLEASE someone HELP.
Here is the protocol I use for TRAP staining (From Sigma)
Fix the cells (4% PFA) for 15 min
Wash twice
Add trap solution (1.8 mL deionzed water, 20uL naphtalon, 80uL Acetate, 40uL Tartrate + 20uL sodium azide, 20uL Fast Garnet)
Incubate 1h at 37C
wash with pbs and evaluate under microscope
Check the pH of your solutions and the expiration date on the reagents..
Thank you Carole, expiration date of the reagents is in April. I will also check the pH of the solutions. Thank you.
Also, ask yourself if you have changed anything such as the source of water used.
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