I'm trying to follow the methodology in one paper that describes the staining of cardiomyocytes with anti-connexin 43. After the procedure for staining, the paper states that "Samples we're alternatively stained with Alexa Fluor 488-conjugated anti-myosin heavy chain to visualize sarcomeric actin; all scaffolds were counterstained with DAPI to visualize cell nuclei". Does this mean that each scaffold is stained with anti-connexin 43, anti-MHC and DAPI, or is it implying 2 sets (anti-connexin 43 & DAPI) and (anti-MHC & DAPI)?
http://www.ncbi.nlm.nih.gov/pubmed/22979989
From the info you've given - it seems to me to be a co-labelled section- and that the second primary antibody was (obviously) conjugated to the fluorophore; whereas the first (anti-connexin 43) was 'separately' labelled with it's specific secondary fluorophore. And from looking briefly at the manuscript (figure 6)- I would interpret this as all staining done on the one section/scaffold.
What I should of said- is that I think the authors are using the word "alternatively" when they should probably have said "subsequently stained with conjugated MHC"
Good luck!
It is possible to strip a Western blotted membrane of a first set of antibodies, and re-probe with a different set of antibodies targeting a second set of antigens (so that the interpretation of the second set isn't obscured by the first.) Is it possible this is what was meant?
Yes exactly Stephanie, I think these transition words really do matter because they make all the difference in understanding the methodology. That was exactly why I was confused. Thank you for clarifying.
Thank you Milliaw, I am not yet however familiar with Western blotting. I was looking at this methodology for immunohistology specifically. But perhaps you are correct as well.
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