I am trying to visualize Nemaline Rods in Skeletal Muscle fiber cross sections. I have read about 10 different gomori protocols, and the protocol seems very straightforward. Every paper I read on it uses these protocols and don't report anything special. I have tried them all, but the staining is never consistent. Sometimes, the fibers are nicely green and have red Nuclei/rods (as it should be), but most of the time, the fibers are stained bright red or purple and the staining is unusable. Is there something I need to look out for specifically doing this staining?
Dear Leander Vonk, pardon me:
GOOGLE-searching for ONLY "modified Gomori Trichrome (LG)" reveals a lot of commercial dye vendors (kits, dye powder etc.)... IMHO you need to - at least - specify WHICH particular >Staining Method< out of the presumably at least 10 different "modified Gomori Protocols" you are using to intermittently get different, hence unexpected staining patterns. You should also tell the interested researcher / colleague, which further/additional histochemical reactions in parallel you usually - on a basic routine - will perform - just to evaluate your findings (e.g. MGT + SDH-COX-stain) ... Also, you might have a closer look into other possible stains / staining techniques (comparing stained section images - e.g.: https://www.google.com/search?client=firefox-b-d&q=modified+Gomori+Trichrome+LG#vhid=6u9LL6s1HG8XmM&vssid=l),. If you decide to reply to my post/comment, you might also - please - add an information which sections you are staining: 'wax' embedded or cryosections ?? and also if there might have been variations in the primary fixation (if chemical fixation was applied initially).
Thank you, with my best regards, W. H. M.
Dear Wolfgang H. Muss , Thank you for the Reply.
Let me specify: I am using Cross-sectional cryosections of snap frozen mouse skeletal muscle at a thickness of 5-10 um. I do not use any fixative on the sections. Here are the Gomori Trichrome formulations i have tried:
1. Commercially available Gomori Trichrome (LG) from Sigma (specifies 0.1% Fast green concentration)
2. My own solution of Gomori Trichrome (containing 0.6% Chromotrope 2R and 0.3% Fast green)
3. My own solution of W. Garvey's Modified Gomori trichrome (containing 0.12% Chromotripe 2R and 0.015% Fast green)
I have tested these solutions at their native pH (2.4), and a raised pH of 3.4 according to Engel & Cunningham's modification
I have also tried chemical fixation using 4% PFA before staining.
The problem remains that sections are not showing a consistent coloring. The expected result should be as follows: Green-blue muscle fibers, Red nuclei and nemaline rods, Green connective tissue.
What happens most often is that the muscle fibers stain completely red and no blue-green is visible. This happens both in fresh sections and in sections that have been stored at -70C.
The protocol listed in Muscle Biopsy: a practical approach states that differentiation using 0.2% (2% in Garvey's modification; I have tried both) should reduce an overly red color. This does have any effect when I try it.
Most publications that show this staining for nemaline myopathies refer back to the Garvey protocol. Sadly I am still not having any luck when trying any of the many modifications. I hope you might have an idea what is going wrong.
Thanks in advance.
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