[Histonet] IF sections look rumpled, in/out of focus

mpkrause <@t> yorku.ca mpkrause <@t> yorku.ca
Thu Mar 20 09:40:22 CDT 2008

I'm not sure if I can get a question posted this way but here's a try:
When I do immunofluorescence for adiponectin in mouse skeletal muscle cross
sections, the section ends up looking very warped and rumpled and is in and out
of focus, almost like it was trying to lift/wash right off the slide.  I've
tried several fixations (4% PFA, Acetone, no fix at all), I've tried putting
them in a 60 C oven for an hour, I've tried air drying, and it's always the
same result.  I tried taking sections and just soaking them in several changes
of PBS to simulate doing the incubation steps in our IF protocol and found the
sections were doing the same thing.  I use gold plus slides, sections are 10um
thick (i'd go thinner but our cryostat does not give consistent thickness below
10um), and come from frozen unfixed muscle.  I've also tried PBS with extra Ca
and Mg, but no luck.

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