[Histonet] Needing advice on mice calvarial paraffin embedding and sectioning.

[Clough, Bret]

Hello Everyone ,
 I am writing you because I need advice on paraffin embedding and sectioning of mice calvarials.  I’ve processed manually a calvarial using the following protocol and had problems with the tissue tearing and or the paraffin detaching (pulling away ) from border with the tissue creating a hole /tear in the paraffin section.  This tissue was processed the follow method:

1.Tissue was fixed in 10% NBF for a couple of days, then placed in Carson’s fix until needed.
2.Tissue washed in 1%PBS x2, placed in 1M EDTA for 7 days to decalcify.
3. Wash tissue in 1%PBS x2, place in 30% ETOH for 30 min at RT.
4. Remove from 30%ETOH and place in 50% ETOH for 30 min at RT.
5. Remove from 50%ETOH and place in 70% ETOH store in 4C refrigerator  until available to continue processing (a couple of days).
6. Remove from 70%ETOH and place in 80% ETOH for 30min at RT.
7. Remove from 80% ETOH and place in 95% ETOH for 30 min at RT.
8. Remove from 95% ETOH and place in 100% ETOH(#1) for 30 min at RT.
9. Remove from 100% ETOH(#1) and place in 100% ETOH(#2) for 30 min at RT.
10. Remove from 100% ETOH (#2) and place in 1:1 (100%ETOH , 100% Xylene) for 30 min at RT.
11. Remove from 1:1 and place in 100% Xylene (#1) for 30 min at RT.
12. Remove from 100% Xylene (#1) and place in 100% Xylene (#2) for 30 min at RT.
13. Remove from 100% Xylene (#2) and place in 1:1 (100% Xylene, 100% paraffin) for 30 min in oven at 59C
14. Remove from 1:1 and place in 100% paraffin (#1) for 30 min in oven at 59C.
15. Remove from 100% paraffin (1#) and placed in 100% paraffin (#2) for 30 min in oven at 59C.
16. Remove and embed.
17. section tissue and look at under microscope.
Please note that the paraffin that I am using is paraffin type 6 from Richard Allan Scientific with a melting point of 55-57c. We also have Histoplast PE from Thermo Scientific but haven’t used it.
Any suggestions on how to solve this problem as well as any protocol one might be willing to share would be greatly appreciated.

Thanks for your help.

 Bret
TAMHSC