[Histonet] B-galactosidase in lung

[t-kuzniar <@t> md.northwestern.edu]

Dear all, 

I am still working on my b-galactosidase in lung tissue, still without
major success. So far, I have tried:
- X-gal reaction on frozen sections, with 0.2% glutaraldehyde fixation
(10 minutes), and detergent washes (protocol below), with or without the
sucrose cryoprotection – I had no signal in the tissue, even though I
know the b-gal is there.
- X-gal reaction on frozen sections, fixed in 100% acetone, with PBS
washes – no signal
- ICH (I use Rabbit IgG alkaline phosphatase VECTASTAIN system) for b-gal 
on paraffin sections, using the polyclonal Ab from Chemicon (has anybody
used it and have comments on it?) – in 0.2% glutaraldehyde, with or
without sucrose cryoprotection step – there was a non-specific staining
of the primary antibody (my “no primary antibody” controls were negative, 
but my naïve animals that SHOULD NOT have b-gal had an intense staining
from this polyclonal Ab)
- ICH for b-gal on frozen sections, with the same antibody and with
either 0.2% glutaraldehyde or 100% acetone fixation – I had an intense
staining of all the controls in my biotin-avidin system.

Protocol for X-gal staining
Sacrifice an animal by injecting an overdose of Nembutal (more than 60
mg/kg) ip
Create a pneumothorax and remove en bloc trachea and heart-lungs. Keep
everything on ice. 
Fill lungs via a cannula inserted into the trachea with ice cold PBS (x2)
Slice lobes by halves, in case of the left lobe – slice in four
Cryoprotect with 15% sucrose in PBS for 1 hour at 4oC
Cryoprotect with 30% sucrose in PBS overnight at 4oC

Cover samples with Tissue-TEK
Freeze in -80oC for >1 hour
Cut frozen sections. 

Fix in 0.2% glutaraldehyde, 5mM EGTA, 2mM MgCl2 in PBS on ice for 10 minutes
Wash in 0.01% sodium deoxycholate, 0.02% Triton X-100, 2 mM MgCl2 in PBS
on ice for 10 minutes (x2).
Wash in 0.01% sodium deoxycholate, 0.02% Triton X-100, 2 mM MgCl2 in PBS
at RT for 10 minutes (x2).
Incubate overnight with lacZ staining solution at 37oC protected from light.

Wash twice in PBS plus 2 mM MgCl2 at room temperature for 5 minutes each
Rinse in distilled water
Counterstain for 30 seconds in Nuclear Fast Red
Wash three times in distilled water
Dehydrate through ethanol (5 minutes each in 50%, 70%, and 100% ethanol)
Clear sections twice for 5 minutes each in xylene
Mount


Thanks, 

Tom Kuzniar