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

[Merced Leiker]

Hi,

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 
(epifluorescent).

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