Beta-gal staining - Please help!!!! (Apr/13/2005 )

Hello fellow lab rats!

I am attempting to stain paraffin-embedded mouse brain sections for beta-gal to no avail. Every protocol I have seen lists this stain for frozen sections. I believe you probably cannot stain the paraffin-embedded sections for b-gal (loss of enzymatic activity due to embedding process), but was told to try anyway....does anyone know if it is possible, and if so any recommendations?

My current protocol:

Deparaffinize:
5 min xylene x 2
5 min 100% EtOH x 2
5 min 95% EtOH
5 min 70% EtOH
5 min PBS

Apply X-gal solution (5mM Potassium Ferricyanide Crystalline; 5mM Potassium Ferricyanide Trihydrate; 2mM Magnesium Chloride
in 1 X PBS with 1 mg/mL x-gal) to sections and incubate at 37°C for 30 min to 24h (check sections under microscope every 2h for development of blue staining)
Rinse with PBS
Wash 2x2 min with diH2O
Counterstain 3 min with Nuclear Fast Red
Wash 2x2 min with diH2O
Coverslip and gel mount

So far, has been in x-gal solution for 29h with no blue developing....
Thanks for the input

Here is my protocol that works wonderfully on cells and whole embryos.
I'm not sure if it will work on sections, but I wouldn't see why not.
Worth a try?

5mM potassium ferricyanide (41mg)
5mM potassium ferrocyanide (53mg)
2mM magnesium chloride (50uL 1M Stock)

Dissolve 25mg X-Gal (40mg/mL) in 625uL N-N Dimethyl Formamide
Add this to the mixture above and then bring the final volume up to 25mL
with 1X PBS, pH 7.6

Filter and add stain to slide
Incubate: for cells ~3 hours
for embryos ~2 hours
for slides ?

Rinse in 1X PBS pH 7.6

Remember it is light sensitive, so keep the stain wrapped in tinfoil and the slides
away from light.

Hope this helps
REmerson

Thanks REmerson. One quick question: how are your cells & embryos fixed?

I fix for 1-2 hours on ice, depending on the size of the embryos.
Here is the formula for the fixative I use:

2.7 mL 37% Formaldehyde (2% Final Vol.)
400uL 25% Glutaraldehyde (0.2% Final Vol.)
Bring it up to 50mL with 1X PBS pH7.6


Hope this helps
REmerson