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connective tissue autofluorescence

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#1 ek24



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Posted 08 September 2003 - 12:08 AM

My job is to find a way to stain paraffin embedded tissues in Hematoxilin & Eosin, scan the slide (automated machine), destain it and then do Fish stain. That way I can compare the morphology of the cells\tissue it’s cytogenetics.
After solving almost all of my chemical properties problems (sodium thiocyanate is still missing) I’ve started performing the stains.
After I destain the H&E I apply DAPI (as for now). I encountered a green background in the fluorescence microscope and I think it’s the connective tissue autofluorescence.
I read that fixing with formaldehyde may cause autofluorescence with amine groups.
DAPI has one. So I tried the DAPI stain fixing only with ethanols (not formaldehyde).
Still, the green background appeared.
My guess is that I didn’t destain the eosin good enough.
Do you know a way of destainin eosin ( pink colored-1% in aqueous solution)?

#2 Victor



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Posted 16 October 2003 - 12:28 PM

A long time ago, I read the eosin is fluorescent on the green channel. I don't think you can circumvent eosin fluorescence and you should avoid it altogether if possible.

Why not try H&E on one section followed by FISH on the serial section? Or, do your sections have to be thick enough to contain whole nuclei for FISH?

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