Using Methyl Green as a counterstain on DAB chromogen stains; IBA1 and GFAP. The Methyl Green solution is 0.125% VWR brand powder, made in 0.1M Sodium Acetate buffer at pH ~.4.2-4.3. The tissue is mounted on slides the night before and allowed to dehydrate. The following day the tissue is rehydrated in distilled water for 2 minutes, then a 5 minute incubation in Methyl Green solution, followed by 15 dips into 0.5% acetic acid in acetone. The slides then go into a series of increased ethanol concentration dehydration steps and coverslipped. The stain appears to be more nuclear in the cortex, and becomes more cytoplasmic in the DG of the hippocampus and thalamic region. I would greatly appreciate input on how to improve the counterstain whether it be changes in solution concentrations, incubation times, etc. I have provided images of the GFAP as well as IBA1 stains, if I can provide anymore information please let me know. Thank you.
Hi James,
I think this is a concentration issue, but I'm not sure. To my knowledge, when methyl green is used as a brightfield staining, it is normally used in conjunction with pyronin y (see https://www.tandfonline.com/doi/abs/10.1080/10520290312120006?journalCode=ibih20). The idea is that one is more specific to DNA, but not completely, and the other one more cytoplasm and when you employ both in parallel, the cytoplasmic staining of methyl green will be prevented or at least masked by the other dye.
I use methyl green sometimes as a cheap fluorescent nuclei stain in cleared samples (if you're interested https://www.nature.com/articles/s41598-019-47336-9 :P). Got the idea for that from this paper: Article A fast, low cost, and highly efficient fluorescent DNA label...
. In the fluorescent staining, the concentration used is much lower, but even there I sometimes see staining of cytoplasm when the concentration is too high.So I guess my advice would be just do a classic methyl green-pyronin y staining, but my experience with the dye as a brightfield staining is very limited.
Hope this helps or someone with more experience can comment! :P
Best,
Sven
Simple issue - ethanol dissolves the methyl green. Unless you want to fade it, work quickly with the ethanol and get it to xylene. Alternatively, you can just clear with xylene and then mount/coverslip.
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