0
Couldn't find any protocols online or at the library either. We've been fixing them in PFA and storing them at 4 deg for now. I've been freezing the rest of the muscles we want in isopentane and cryosectioning them.Here's one paper I found but maybe I'm a total newb on this one so does anyone have any more details? Do you leave them whole for the perm/block/Ab? Would the antibodies be able to penetrate in a whole TA? Can you then just section them afterwards or do you isolate fibers as they say? I'm also just curious as to why we don't just cryosection them like the rest of the skeletal muscles?
Thanks a lot if anyone can help out.
from Méjat 2009 "Lamin A/C–mediated neuromuscular junction defects in Emery-Dreifuss muscular dystrophy":
For immunofluorescence microscopy on mouse muscle fibers, tibialis anterior muscles were dissected and fixed in 4% PFA/PBS for 30 min at RT, rinsed with PBS, pH 7.5, at RT, and permeabilized and blocked in the blocking buffer (1% BSA/0.5% Triton X-100/PBS) overnight at 4°C. They were then incubated overnight at 4°C with the primary antibody (1:500) in blocking buffer. Samples were washed three times for 1 h each time in 0.5% Triton X-100/PBS, incubated with secondary antibody (Alexa Fluor 568 goat anti–rabbit, 1:500; Invitrogen) and Bgt–Alexa Fluor conjugate (1:1,000) for 1 h at RT in blocking buffer, and then washed three times for 1 h each time in 0.5% Triton X-100/PBS, plus an overnight incubation at 4°C. Finally, isolated fibers were flat-mounted in mounting medium containing DAPI. Z serial images were collected using a PlanApo 100× 1.4 lens (Nikon) on an Eclipse E800 (Nikon), and image stacks were projected into a single image using Metamorph 6.3 software (MDS Analytical Technologies). A minimum of 100 fibers from at least three different mice of each genotype were analyzed for each staining.
