[Histonet] Re: LacZ and decalcification

From:"Johnson, Teri"

Nicole, wow! What a well informed question, well done!

I don't know everything there is to know about B-gal and x-gal staining. I can only give you advice based on my experience. You may well have more of it than me.

Having said all that, we've tried to stay close to the original x-gal protocol we saw from Lobe etal. using Z/EG mice. Their fixative was 0.2% glutaraldehyde in PBS buffer with EGTA and MgCl added. Larger samples were fixed in 0.2% glutaraldehyde with 2% PFA in the above buffer. All were fixed on ice, with shaking. Fixation was minimal, less than 4 hours for the largest samples.

We have never tried to do x-gal staining on paraffin embedded samples. We only do them on cryosections and whole mounts. If we do whole mounts, we post-fix overnight (or longer depending on the sample), and do paraffin sections (8 microns thick) to demonstrate the activity.

You have every right to be worried about bone samples, decalcification, and fixation. We have approached these samples in two ways and have had success with both.

One technique is from a journal article in J Bone and Mineral Research, Vol. 20, Num. 7, 2005 (Hens, etal). Here is their protocol (copy/pasted verbatim from the article), which we followed except we used 10% EDTA. The results were good, and the decal only took 3 days (we checked it with faxitron). Your mileage may vary depending on the age, strain, and genetic background of your mice.

>>Adult bones were
fixed in 2% paraformaldehyde and 0.02% glutaraldehyde in
PBS for 1 h at room temperature and washed twice in PBS.
Bones were first decalcified in 4% EDTA for 17 days and
then washed in PBS for 3 h before being incubated in 0.1%
4-chloro-5-bromo-3-indolyl -D-galactopyranoside (X-gal),
2 mM MgCl2, 5 mM EGTA, 0.02% Nonidet P-40, 5 mM
K3Fe(CN)6, and 5 mM K4Fe(CN)63 H2O at 30C overnight.
Bones were subsequently washed once with PBS and
then postfixed in 4% paraformaldehyde at 4C overnight.
Individual bones were rinsed in 70% ethanol, embedded in
paraffin wax, sectioned, and counterstained with eosin.<<

You'll notice the gluaraldehyde concentration is very low. I can't imagine it has that much effect since it takes so long to penetrate tissues. They do cut their bones in half to allow better penetration. You will also notice they fix at room temperature (my heroes!).

The next technique involves whole mount staining first, and then post-fixation and decal in formic acid (Immunocal). When we first tried using EDTA post-staining, all the blue stain dissolved. We found that if we use formic acid instead, it preserves the x-gal staining. Again, the bones are opened (in the middle if interested in the ends of the bone, or in one end of the bone if interested in the middle) and fixed for a couple hours, then rinsed, then stained overnight with x-gal reagent. Post-fixed, decalcified, then paraffin processed, sectioned and counterstained with Nuclear Fast Red.

I hope this helps!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

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