[Histonet] RE: Double skeletal stain on archived tissues

[Johnson, Teri]

I agree that it's possible your fixative has become acidic after one year's time and could be decalcifying your specimen.  We did a simultaneous study using one adult mouse that had been fixed for over a year in 10% neutral buffered formalin, and one that had been freshly euthanized and processed according to our recommended protocol.  We achieved staining on both, but the freshly processed sample was much more intense with much better quality than the previously fixed sample.  Actually the archived tissue had very poor staining, nothing that was publishable or usable after we finished the technique.  

We have found that if the animal was placed and stored in a freezer soon after deat, there seems to be little to no compromise of the staining of these samples.  We have not tested for long-term freezer storage however.

We have not run into the problem of stain penetration on any of our mouse samples.  We do remove the fat pad on the back of the neck on mice as part of the dissection and evisceration, and will also use an 8 hour - 1 day soak in acetone before starting the staining procedure.

If you have access to an x-ray machine or faxitron, perhaps you can see if specimen decalcification is the problem here.

Teri Johnson
Stowers Institute for Medical Research
Kansas City, MO