We have injected GFP and td-tomato viral tracing injection in the mice brains to see axonal projections, and we are using tertiary butyl alcohol instead of ethanol for the dehydration process as well as xylene. But we are losing our fluorescence.
What we can do please suggest any other solution or any procedure.
So please, Thank you in advance.
I hope this will help us.
Note: We don't have cryostat with us.
Hi Avinash,
Am I assuming correctly that you are cutting paraffin sections from the mouse brains? The processing is going to degrade the native fluorescence in the sample. However, the fluorescent protein can be detected with antibodies after deparaffinization.
I hope this helps.
Best,
Simone
Simone Schindler Thank you for your insight, yes you are right we are doing it in the paraffin section. But we referred to a paper called "Maintenance of Fluorescence During Paraffin Embedding of Fluorescent Protein-Labeled Specimens". They have done the same process and they got the fluorescence. It is not happening with us. So is there any other alternative or process that can help us? Please let me know if there is any other mechanism that affects this.
For optimal fluorescence, try not to dehydrate your tissues. Use alternative protocol without dehydration and use aqueous mounting media.
Louis-Charles Béland Thank you Dr. Louis for your suggestion. Then I think I have to go with the cryostat method right? But Sir, is there any other way without dehydration because I cannot find any.
Ok, thank you sir.
Avinash Wadkar , The method you use to cut your tissues doesn't really affect your fluorescence. Once they are cut and mounted only microscope slide, you can deparafinize and rehydrate before regular immunofluorescence staining. Do do not dehydrate after the IF staining and mount in a fluoromount medium (aqueous).
If you really cannot keep your fluorescence in any way or my previous proposed method doesn't fit your needs, use an immunohistochemistry protocol with DAB, using against your GFP or tdTomato and dehydrate at the end as you usually do.
Louis-Charles Béland Sir, For the paraffin embedding system we have to dehydrate the tissue before staining but here we have already injected the viral tracer in the brain so no need to have a staining process.
I think I have to go for the cryostat method, and aqueous mounting media like glycerol.
Thank you sir for your valuable insights.
If you are working with mouse brain, would it be possible to instead fix them using 4% paraformaldehyde pH7.4? It helps retain endogenous fluorescence compared to paraffin embedded tissue.
I am fixing them in PFA only, but we used a paraffin embedding system to take sections. Post-fixing in PFA we have to dehydrate the tissue and incorporate wax into it. For sectioning, we can't use PFA fixing alone.
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