Hi Valeria. I do a lot of mouse IHC (FFPE and frozen sections), and
mouse tissue, *especially* ovary, is highly autofluorescent, so you have
my sympathies. In fact, when I look at a fresh mouse ovary under the
fluorescent dissecting microscope, it really lights up! One of the
things I have found rather helpful in reducing the amount of
autofluorescence is Sudan Black.
So, you're supposed to do the autofluorescence quenching step after
finishing the immunofluorescence stain and I have tried this, and it
*does* work; however, I have had a couple of antibodies
(anti-cytokeratin, for example) that don't like this treatment, and so I
do the quenching after the blocking step. I would try both ways to see
which one works best.
I have tried different quenching times on different mouse tissues (not
ovary, unfortunately) and have found that anywhere from 10-30 minutes is
required. After quenching, wash the slide well with squirts of wash
buffer to remove precipitates...I usually use a regular, plastic
transfer pipette for this. Follow the squirting wash with a 10-minute
wash in wash buffer. Then, either coverslip if you've finished your
staining, or continue with your primary antibody incubation.
Here is the protocol for preparing the Sudan Black solution:
Make up 0.l% solution in 70% ethanol. Heat to boiling, then cool and
filter.
I think that's about it. Best of luck,
Jacqui Detmar
Samuel Lunenfeld Research Institute,
Mount Sinai Hospital
600 University Avenue
Toronto, ON, Canada
M5G 1X5
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