[Histonet] Question about De-calcifying mouse paws

[Gayle Callis]

Jamie,

Well you are NOT alone in this, you have described a common problem we 
experienced also.  Liz Chilpala should also have input on this.

Mouse paws with ankles are really dense (tightly packed tiny bones and 
connective tissues).  We fix paws much longer than 24 hours due to their 
density, often up to a week. Have you thought of perfusion to help with 
speeding up fixation?

Decalcification should be done in the time frame you gave.  Did you look to 
see what the concentration of formic is in your CalRite MSDS?  I think 
maybe as high as 10% but if concentration of methanol is high, then 
decalcification slows down.  We have used 70% alcohol to stop 
decalcification since calcium does not ionize well in presence of bipolar 
alcohols.  Use of alcohol, historically, slows down decalcification rate, 
hence damaging effects of mineral acids on tissues (called 
overdecalcification, although overexposure may be a better term) , etc i.e 
nitric formulations.  Perenyi's decalcifying solution an example

If bone is totally fixed, we prefer 10 to 15 % formic acid from 90% formic 
acid stock (V/V) and paws can still take a couple of days.  I do a weight 
gain/weight loss endpoint check and since most samples are the same size, I 
work with several from both the control and OA induced group for endpoint 
testing.  Rather than weigh each and every sample in a group of 50 mice!!

You did not say how long you process, but the chalky, opaque look can be 
caused by

1. Underdecalcification (endpoint test helps unless you have an xray 
machine or microCT scanner, even better)

2. Poor dehydration/clearing (increase times in all stations - I do 2 hours 
per station in a VIP and use Tissue Prep 2 (harder) for infiltration and 
embedding) but infiltrate at 58 to 60C with this, 3 to 4 changes always 
using vacuum/pressure/ambient temps for processing.

3.  OR both of the above.  When that happens, heavy sigh and see what has 
been done, then correct accordingly.

You can try and melt down the blocks, go back into vacuum/paraffin for a 
few hours - 2 or so, reembed and see it that improves things. If tissue 
swells away from trimmed blocks after soak, back off - warm water followed 
by ice cold water often helps.  Oversoaking only exacerbates the problem!!

I also know of another decalcifier that works faster and with cartilage. It 
may be more damaging to IHC though but works with cartilage stains and 
H&E's - I will be happy to send recipe to you.

Good luck

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)