RE: Lac-Z labeling in bone specimen

Are you using rats or mice? For Lac Z reporters, you need to:

(1) Fix in formalin overnight (12-24 hours)

(2) Rinse with water

(3) Wash with the following buffer, 3 times for 5 minutes: X-Gal buffer =

0.1M phosphate, pH 8.3-8.5, 2 mM MgCl2, 0.1 % sodium deoxycholate, 0.2 % Triton X-100 (or Nonidet P-40)

(4) Make your x-gal (5-bromo-4-chloro-3-indolyl-B-d-galactoside) stock solution 40 mg/mL in DMSO (dimethylsulfoxide). For example: purchase 100 mg of X-Gal prepare 40 mg/mL solution by adding 2.5 mL of DMSO.

(5) Make the following: 50 mM K3Fe(CN)6 (in distilled water) and 50 mM K4Fe(CN)6-3H2O (in distilled water)

(6) Make the working x-gal stain as follows: Place 70 mL of X-Gal buffer (as prepared in # 3 above) into a 100 mL graduated cyclinder, add 10 mL of 50 mM Fe3(CN)6, add 10 mL of 50 mM Fe4(CN)6-3H2O, add 2.5 mL of the 40 mg/mL x-gal, add more x-gal buffer to a final volume of 100 mL.

(7) Place specimens in the stain over night (12 - 24 hours).

(8) rinse with water

(9) Place in formalin to complete fixation (2-3 days for mouse legs, 4-5 days for rats).

(10) Decalcify. We use 5 % formic acid in distilled water. We discard the used formic solution and replace with fresh formic every morning. On the morning of the fourth day of decalcification, we will check the used formic solution for dissolved calcium by adding 5 mL of this solution to a test tube that contains 5 mL of 5% ammonium oxalate. If a white precipitate forms, the used formic solution contains calcium. That means there is still calcium in the specimen and we continue decalicification with fresh formic solution. Check every morning for calcium in the used formic solution. When no white precipitate forms in the 5 % ammonium oxalate solution the decalcification is complete.

(11) After decalcification is complete, rinse the specimens with water. Then process for paraffin as usual.

After processing, cut sections, place on slides, deparaffinize to distilled water, stain in 0.1 % saffranin O for 1 - 1.5 minutes, rinse with water, dehydrate through serial alcohols, clear with three changes of xylene and coverslip with permanent mounting media.

LacZ expression will appear bright blue-green

All other structures will be in shades of pink and red.

Hope that helps, Donna Montague

University of Arkansas for Medical Sciences

Department of Physiology & Biophysics and

Center for Orthopaedic Research

4301 W. Markham St. # 505

Little Rock, AR 72205

Dear Dr. Montague,

I am a reserch fellow in Dept. of Orthopaedic Surgery, National University of Singapore. I read this answer from you. May I have more suggestion from you as my specimen are bone which need to be decalcified. Normally I fix the specimen (knee joint ) with formalin for one week, and decalcified for two weeks, and dehydrate and paraffin. From your email I understand that I can do the x-gal stainin first. I am wondering whether we do staining in fixation step or after decalcification?

Appreciate your help,

Thank you very much,

Li Li

MontagueDonnaC@uams.edu

You can use either. If you decide to use GFP use one of the enhanced
variants, e.g. EGFP from Clontech. This fluorochrome is stable in to
paraffin processing. LacZ is also stable after the X-gal precipitate is
developed. So when using Lac Z, formalin fix stain in x-gal then process for
paraffin. Hope this helps, Donna Montague, Center for Orthopaedic Research,
UAMS, Little ROck, AR

-----Original Message-----
From: Garry Ashton [mailto:GAshton@picr.man.ac.uk]
Sent: Wednesday, May 23, 2001 8:45 AM
To: 'histonet'
Subject: GFP / LacZ


Dear histonetters,
a researcher asked me for the relative merits / disadvantages of GFP against
Lac Z  in histological terms as a reporter of gene expression in paraffin
sections.
Initially he doesn't want to use immunocytochemistry.
Can anybody give any constuctive advice.
Many thanks
Garry Ashton

Paterson Institute
Manchester
UK