[Histonet] regarding problems with decalcification of rat maxilla's

[smit dangaria]

Hi,
I am a graduate student at University of Illinois, chicago.  I have been having
tremendous problems with sectioning of paraffin embedded rat maxilla with molars and I
searched the Histonet archives and found that people here have invaluable
experience, insight and suggestions for various problems. 
Heres my problem. :
As I section the block the section would crumble and tear and has a dusty
dry look to it and if I run my fingers over the block I can clean off the
dusty apperance of the block. (does that mean it wasnt decalcified well, or paraffin infiltration wasnt enough? or is it possible there is some EDTA salts or left over paraformaldehyde but I did wash the samples with water after being in paraformaldehyde and EDTA )  After decalcification the samples felt very
soft and I could bend the maxilla and were flexible and could be cut with
an ordinary blade.

Here is the protocol I used for decalcifying the samples and the
processing details. I would really appreciate if you guys can give me some
suggestions as to what I can do about these samples and what must have
went worng.

Here's the method that I used for rat maxilla's with teeth in them.
1. Fixed in 4% paraformaldehyde for 4 days and then washed with distilled
water.
2. Decalcified using 8% EDTA with change to fresh solution everyday for 3
weeks using the microwave technique. (eg: the maxilla from one rat were
decalcified in 40 mL EDTA)
3. After decalcification the samples were washed in distilled water for
couple of hours with fresh changes every 30 mins.
4. The samples were then transferred to 50% ethanol for an hour and then
70% ethanol for an hour and then overnight in 70% ethanol.
5. Then 95% ethanol for 1.5 hours with fresh changes every 30 mins
6. Then 100% ethanol for 1.5 hours with fresh changes every 30 mins
7. Then half 100% ethanol half 100% xylene for 20 mins
8. Then Xylene for 3 hours with fresh change every 45 mins (samples could very clear after this step).
9. Half Xylene half paraffin for 30 mins.
10. 100% paraffin (paraplast plus) for 3 hours with fresh change every 45
mins and then embedded in paraffin.

I would really appreciate if you guys would guide me as to what I can do so
that I can use these samples to cut sections. Thankyou for your time and
help.
Sincerely,
Smit.


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