[Histonet] IF muscle cytoskeletal proteins - interior of myocytes doesn't stain

From:Merced Leiker


I'm trying to stain hamster skeletal and cardiac muscle for Troponin  I, 
Troponin T, and Myosin Heavy Chain (separately). When I do Z stack imaging 
at 0.5um steps, only the outermost myofibrils (near the sarcolemma) of most 
of the myocytes are stained; the interior (particularly of te larger 
myocytes) is unstained, looking like a gaping black hole.

The tissues were snap frozen, cut 8um thick, fixed on the slide in 4%PFA, 
and permeabilized in 1% Triton.  All antibody incubations were done at 
either 1 hr RT or overnight 4oC.  The microscope used was Zeiss Axioimager 

Can you tell me if this is expected staining for these material and 
conditions, or if something is wrong?

Thank you.

Merced M Leiker
Research Technician II
354 BRB (Lee Lab) / 140 Farber Hall (mail)
School of Medicine and Biomedical Sciences
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214
Ph: (716) 829-6033
Fx: (716) 829-2725

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