10X PBS washing.... - (Mar/07/2007 )

Dear all,

I made a terrible mistake. When i pre-treated my parafin-bedded brain sections for my in situ hybridization, at each step i washed my section with 10X PBS buffer, instead of 1X.... i know this sounds silly but it actually happened, for this is my first time to do it.

Does that matter? i presume this is just washing, nothing big enough to destroy mRNA. But i am not sure.

Please let me know if you have some ideas. thanks a lot

ive never tried it myself but apart from your sections being a little saltier than usual i doubt if it will be that much of a problem - the question is tho did you use the same pbs to dilute any antibodies etc which you then put on the tissue.

yeah, i used the same 10X PBS for fixation and washing etc. But when i found this mistake at final stage, I changed to normal 1X PBS for washing several times... i am not doing immunohistochemistry but in situ hybridization, with radio-labeled riboprobe.

Tomorrow i will do hybridization with my riboprobe, and hopefully see what happens.

anyway, i am a bit relieved, thanks a lot.

sorry force of habit i have immuno on the brain - hope it works out