c2c12 cells labelling questions AGAIN!!

Qunfeng Dong qfdong at iastate.edu
Sun Nov 1 01:22:24 EST 1998

Hi there:

  Several days ago, I posted a question about c2c12 cell fixation questions.
I thank all those warm-hearted people for their kind answer. I will have a
post later regarding all those people's answers. However, I am still having
trouble to get very specific labeling. I am trying to label myosin using
MF20. I know I can see the stress-fiber like structure and myofibril inside
those myotubes. However, my labeling seems mostly label the whole myotubes.
I tried both 2% and 4% paraformaldehyde fixation. I tried different
concentrations of TritonX-100 to permeabilize cells. I tried both 1% BSA or
5% Goat serum for blocking. I tried different wash time (from 30 sec to 5
mins each). I even added 0.01% TritonX-100 to the wash buffer(PBS). I am
still getting background. I know my antibody is good which specifically
labels chicken or beef myofibrils. 

  So, any more suggestions?

  Some people think washing for about 30 sec each time is enough, some
people think the total washing time should be equal to the total Ab 
incubation time with each washing more than 5 mins. Anyway, I have a feeling
that I have a problem with the washing step (or blocking). 

  It's just getting frustrating. Any suggestion is welcome! (Please send me
an personal email, I can write to but can't read this newsgroup recently :(((

Qunfeng Dong
qfdong at iastate.edu

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