[Histonet] Loss if LacZ staining after mounting slides,

[Xuesong Chen]

Hi histonetters,

I need help on lacZ counter staining.

I’m trying to stain bone/cartilage for beta galactosidase. H&E does not seem a
good choice because lacZ blue is not accentrated. I have been using Nuclear
Fast Red (CI 60760) as a nuclear counterstain, but after dehydrating and
mounting I lose the the entire beta gal over the course of two or three weeks.

My protocol:

Cryojane sectioning femur/tibia and air dry the slides
10 min in 1XPBS
5 min in 2%PFA in 1XPBS
10 min washing in 1XPBS
Beta galactosidase staining overnight at 37C in dark
10 min washing in 1XPBS
15 min counterstaining in 0.1% nuclear fast red
10 min washing in 1XPBS
5 min in 70% ethanol
5 min in 95% ethanol
5 min in 100% ethanol
5 min in Histoclear
5 min in Histoclear
Cytoseal 60 (low viscosity) to cover the slip.

I'm wondering is there any people using nuclear fast red for beta-gal 
counterstain and whether you have experienced the same problem, or
alternatively, advice any possibilities that cause this.

Thanks

Xuesong