[Histonet] Ovary aromatase

[Jacqui Detmar]

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