When I performed Hoechst staining or HE staining in muscle fiber (Gastro muscle), it could be noticed very frequently that some fiber contained holes in the middle. Cyrosection was used in that detection. So what is the possible reasons leading to the phenomenon? Thanks for your suggestion!
Looks like the muscles thawed at some point during the process. Are you extra sure you always kept them at low temperatures for every step of the process, including transport and cutting?
Dear Xuerong,
it looks like the freezing procedure was not adequate. You could try to shock freeze your tissue in liquid isopentane cooled by liquid nitrogen.
Best regards,
Daniela
Thanks Daniela and Silva! Your commments are very helpful! Daniela Rodler Silvia Torchio
Actually I fixed the muscle using PFA at 4 degree for 2 hours, then tranferred the tissues to 30% sucrose to incuate at 4 degree for overnight. After that, they were embedded with OCT.
The tissues express Tomato fluoresence, so I want to fix them in case the fluresence disappear. So could I omit the sucrose procedure to reduce possible muscle thawing since it takes a long time? It seems you don't adopt dehydration procedure if freezing the tissue directly.
Thanks for your suggestion!
Best regard
Xuerong
I'd go with the protocol suggested by Daniela Rodler : basically, after dissection, each muscle is placed on a cork support with a small amount of inclusion gum, then immersed in nitrogen cooled Isopentan, then in liquid nitrogen and stored at -80°C. WOrks well for me!