Hello everyone,
In order to cut 10 µm slices of mammospheres with cryostat, I had to colour them with eosin to be able to follow them while cutting.What a bad idea you'll tell me, because eosin is fluorescent!
But now I want to process immunofluorescence staining with this precious material, and I can't because it seems impossible to get rid of the fluorescence of the eosin. I tried to wash the slides with PBS or alcohol (70°), and nothing works.
Do you have an idea ? I'd like not to do the preparation again..
Thanks beforehand,
CD
We've solved problem of high autofluorescence in liver criosections (not stained with eosin) by short immersing in 96o EtOH and PBS consequently. But morphology suffered from this procedure.
May be series of immersing in good solvents for eosin will help to eliminate it.
In accordance to data from attached links form of eosin could affect solubility so find the best solvent for your form which do not interfere next stain protocol.
SOLUBILITY IN WATER
Soluble (400 g/l)
SOLVENT SOLUBILITY
MEG (850), DEG (600), PG (750), Glycerol (800), Methanol (250), Ethanol (30), Methoxyethanol (120)
My first idea was to use mix of DMSO:EtOH (1:1) but taking into account data listed above EtOH is not the best.
An alternative idea to use protocol with picric acid for criosection https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-frozen-tissue-sections
Please share your experience after all!
Have a nice day!
http://www.chemicalland21.com/specialtychem/finechem/EOSIN%20Y.htm
http://stainsfile.info/StainsFile/dyes/45380.htm
eosin can be dissolved in 90% to 95% alcohol. you try it with 90% ethanol and section can be immersed upto one hour which is sufficient to dissolve eosin.
good luck.
Perhaps you could try avoiding the affected wavelengths using fluorophores like alexa fluor 405 or qdots. You could counterstain nuclei with a far-red shifted and highly fluorescent stain as methyl green. This option would not affect your tissue morphology nor will it require more sections.
Another solution is photobleaching.
https://www.google.ca/webhp?sourceid=chrome-instant&ion=1&espv=2&ie=UTF-8#q=photobleaching+of+autofluorescence+for+immunofluorescence+applications&start=10
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