[Histonet] RE: x-gal staining FFPE

[C.M. van der Loos]

Dear Jenny,
I have optimized X-gal staining (acc. to Bondi et al.) for IHC purposes (FFPE or cryo) last year. The protocol below is what came out best. 
Given that the enzymatic acitivity survived all the steps to make this whole mount(??), this should work for the detecting the lacZ gene as well. 
Good luck !

Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center
Amsterdam - The Netherlands



ß-GALACTOSIDASE activity

using X-GAL and ironphosphate
[Bondi et al. (1982) Histochemistry 76: 153]

Substrates:
• 5-Bromo-4-chloro-3-indolyl-ß-galactopyranoside (= X-GAL from Roche)   
  40 mg/ml in N,N-dimethylformamide, store at -20°C   

Iron-phosphate solution, store at +4C
• Phosphate buffered saline pH 7.4        50 ml
• MgCl2.6 H2O	                          10 mg
• Ka-ferricyanide	                  49.5 mg
• Ka-ferrocyanide	                  63.5 mg

Mix just before use:
Iron-phosphate solution                  1000 µl
X-GAL                                      25 µl
		
• Drop onto specimens and stain for 15 - 60 min at 37°C
• coverslip with either an aqueous-, alcohol- or xylene-based mounting medium

Result X-Gal substrate: turquoise precipitate at ß-GAL activity


----- Original Message ----- 
>From  Jenny Oblander <Jenny-Oblander <@t> omrf.ouhsc.edu> 
Date  Thu, 17 Jun 2004 15:16:50 -0500 
To  "Histonet (E-mail)" <histonet <@t> lists.utsouthwestern.edu> 
Subject  [Histonet] x-gal staining FFPE 
Hi Everyone,
I need your help. My boss wants to do a x-gal on FFPE sections. I
have tried to find a protocol on the web but I can't find anything but
cryosection staining or whole mount. My question is ...Can you stain a FFPE
slide with x-gal? And if you can, how? Thanks Jenny

J.Oblander, HT (A.S.C.P.) 

Comparative Medicine
Oklahoma Medical Research Foundation MS#32 
825 NE 13th St. 
Okc,Ok 73104 
jenny-oblander <@t> omrf.ouhsc.edu 
405-271-7083