I have tried the Ogilvie protocol with prestain buffer at pH 4.3 and 4.5 but the fibers are either all light blue or dark blue respectively. I've come across many papers which claim to use this protocol with mouse tissue and stain all 4 fiber-types, but I don't have any other colors but blue. Am I dehydrating for too long? Can anyone help? I'm interested in changes in the ratios of type II fiber subtypes between experimental conditions. Thanks!
Hi Edlira,
if you are interested in quantifying changes in fibertype, why don't you use antibodies instead? You can buy the 4 antibodies for type 1 (BA-D5), type 2A (SC-71), type 2X (6H1) and type 2B (BF-F3) from Developmental Studies Hybridoma Bank (DSHB): http://dshb.biology.uiowa.edu/ . They're really cheap, and work very well. I made a protocol for such kind of staining, if you're interested. Since three of them belong to different Ig subclasses, having the proper secondary antibodies, you can stain 3 of them at the same time, and eventually add the missing one with an additional "blocking-primary antibody-secondary antibody" step (6H1 and BF-F3 are both IgM). Or add laminin staining, so that you'll also have the fibers outlined, making very easy to count/measure fiber size/type.
Hi Stefano! Thanks so much for your help! Can you send me the protocol at [email protected]? is IF considered as sensitive as the ATPase staining? Or asked another way, if theres a fiber-type switch, would the reviewers accept IF data as opposed to ATPase? I would very much prefer to use antibodies because I have more experience with IF but I was told the enzymatic stain it superior. Thanks so much for your help again!
Sure, I can send you the protocol.
About the sensitivity, I can not 100% guarantee that one reviewer won't ask for ATPase staining... However, I think it will be more likely that someone will ask for a myosin separation gel. At least, this is what happened to us, some years ago.
Moreover, I stayed many years in Stefano Schiaffino's lab, and I can tell you that we NEVER performed a single ATPase staining!
Hi Stefano,
I am currently using the antibodies from DSHB for fibertype staining and although I've had success with SC-71 and BA-F8, I am having trouble with BF-F8. Please could you email me your protocol? My email is [email protected].
Many thanks in advance,
Anna
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