Hi, Melanie.

Perhaps, mounting medium is highly miscible (having glycerol or something else?) with methylene blue.

- Preparation of petroleum jelly (Vaseline®): paraffin wax mixture

Mix Vaseline and paraffin wax (9:1 weight ratio) in a glass beaker. Heat gently until melted, mix well, then suck into 10 ml syringes, cool at 4C until use (link) http://reocities.com/CollegePark/den/1291/protocols/dolly.html

When you make your brain section slide, make 4 columns of pillars with vaseline:Paraffin wax mix in syringe on the glass slide just outsides of the tissue section by gentle pushing plunger. Just 3~5uL solution drop size will be enough, with ~2mm height . Add more methylene blue sol that is centrifuged, taken supernatant om the tissue section. Now put your coverslip on the 4 column pillars without touching the methylene blue yet. With you 21G hyperdermic needle, gently press little by little the coverslip just on the pillar spots diagonally. An then the other diagnal spot with gentle pressure until the coverslip touches methylene You could add more methylene blue by the side between coverslip and glass slide if you lost the solution.

If you use in H&E histology, you will completely lose the color due to many different strong solvents during whole procedure.

Best wishes

Dear Melanie Eve, you haven't indicated anything about the purpose of the marking by injection and (a) fixation of the animals brain(s) after dye injection tracing the way of the needle. Also you haven't said much about how you deposit the dye properly in the brain (tissue) expected to be displayed after i) processing the classical way into paraffin as embedding medium (and therefore cutting paraffin sections expected to display traces of the dye) or ii) freeze the brain tissue and produce frozen sections.

It might be of interest for you to find at least one Q&A thread present already in ResearchGate dealing with dye injection (Pro's and Con's) into brain, cf:

https://www.researchgate.net/post/Long-lasting_non-toxic_dye_injection_in_the_brain? but you might find also more information on the dye methylene blue in general using the search words [in ResearchGate's SEARCH function, cf. rectangle with 'Search' and loupe-icon above FOLLOW/FOLLOWING, upper right menu line):

methylene blue dye injection brain or

methylene blue dye injection brain section mount dye bleeding or

methylene blue vanish dye injection brain mount dye bleeding.

Edited some minute after initial posting for the full text 'Search' in RG system (Q&A as well as authors, publications, etc.) .

Leucomethylene blue is colorless and in contact with oxygen become bliue. With coverslip there is no contact with oxygen from air and MB become leucomethylene blue colorless.