RE: B-Gal stain for paraffin

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Date:Mon, 26 Jul 1999 09:44:56 -0500
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Sorry it took so long to get back to you but we've been busy.
First: Look at the following article. "LacZ Staining in Paraffin-embedded
Tissue Sections" P. Jan Hendrikx et al, J Histochem & Cytochem
44(11):1323-1329, 1996
Second: We've done this procedure a time or two and it works well.
	1. Fix the tissue in 10 % NBF (neutral buffered formalin) for 30
	2. Wash in the following buffer x3 5-10 minutes each:
		0.1 M Na phosphate (pH 8.3 - 8.5)
		2 mM MgCl2
		0.1% Na deoxycholate
		0.02% Triton x-100 (or Nonidet P-40)
	3. Stain overnight in the dark with the following solution:
		1 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-B-D-galactoside;
Fisher or SIGMA)
		5mM K3Fe(CN)6
		5mM K4Fe(CN)6 - 3H2O
	4. Rinse throughly in dH2O then place in 10 % NBF or 70 % ethanol
until properly fixed.
	5. Process as usual.
	6. Cut at 4-6 microns
	7. Deparaffinize to dH2O
	8. stain with 0.1% safranin O (Gayle Callis' suggestion that worked
very well), rinse briefly, dehydrate quickly, clear and permount.
	Blue-green x-gal staining
	Pink-Red background	

Note on #3 above:
	These are final concentrations of the critical reagents. The process
for making this solution is complex and follows.
X-gal: Purchase 100 mg x-gal. Add 4 ml of n,n-dimethylformamide such that
the concentration of x-gal is now 25 mg/mL
Potassium ferricyanide (FW 329.26): Stock solution = 0.25M weigh 8.23 g and
dissolve in 100 ml dH2O
Potassium ferrocyanide (FW 422.39): Stock solution = 0.25M weigh 10.56 g and
dissolve in 100 ml dH2O
In a 100 mL volumetric flask (preferred, 100 mL cylinder okay, add 4 mL (all
the dissolved) x-gal + 2 mL Kferri stock + 2mL Kferro stock and bring to 100
mL with wash solution (#2 above). Mix well. Use immediately.

If yo have further question or need any clarification, feel free to contact
me. Good luck, Donna Montague
UAMS Orthopaedic research
(501) 686-8739

-----Original Message-----
From: Robbin Newlin []
Sent: Tuesday, July 20, 1999 2:09 PM
Subject: B-Gal stain for paraffin

Hi all
Does anyone have a protocol for B-Gal staining of paraffin embedded tissues?

Robbin Newlin HT-ASCP
Manager Histopathology
The Burnham Institute
10901 North Torrey Pines rd.
La Jolla, Ca. 92037

Phone: (619) 455-6480 ext. 3552

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