[Histonet] Re: rat maxilla processing

[Gayle Callis]

Dear Smit You wrote:  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.Reply:  You bone sample is underprocessed, poorly infiltrated with paraffin and may not be totally decalcified.  When you do the fixation, fix longer than 4 days, as rat maxilla is not a small sample and complicated by teeth in situ.   It would be better to perfuse the animal with your PFA solution, then dissect off the maxilla, and immerse into fresh PFA for several days.  If perfused then 4 days may be adequate.  Rinse with running tap water, and immerse into your EDTA (what is pH?) Also, use 20 times the volume of EDTA to size of tissue, and change the EDTA after a couple of days to replenish the EDTA.  EDTA is safe to use over a weekend so you do not have to change it so often.  You need to do endpoint determination to know when the bone is decalcified and I will be happy to send a simple weight loss/weight gain method that works for EDTA or use a FAXITRON  xray determination.  After EDTA, rinse with running  tap water for several hours, not distilled water .  Please contact me for the endpoint determination, I will send as a separate attachment. To process, start the bone in 70% alcohol.  The time changes in each of the alcohols, clearing agent and paraffin are the same.  2 hours per change (30 minutes per change for a total of 1.5 hours is not doing the job!)  If you only have a small portion of the maxilla, you can reduce the time to 1.5 hours per change.  I hope you have a tissue processor to do this for you, as you need to use vacuum and pressure for the best results. 70%  ethanol80% ethanol95% ethanol X 2 changes100% ethanol X 2 changesXylene X 2 changes and to get rid of brittleness, use one change of Richard Allan Clearite 3, then 1 change of xylene.  You can reverse the order of these two clearing agents without problems.  Paraffin X 3 changes (minimum) at times suggested at NO more than 60C.  Heat is an enemy that contributes to hardness of decalcified bone.  To achieve vacuum pressure with hand processing, use a vacuum dessicator, and hopefully a heated vacuum oven for the paraffin steps.  Use a harder paraffin - there are several on the marked.   Tissue Prep 3 is excellent for bone as you want to try and match the hardness of your embedding media to hardness of decalcified bone as much as possible. What is meant by an ordinary blade?  A low profile or high profile disposable blade?  or are you using a c profile steel knife?   High profile blades give much more stability when sectioning bone, and after a very careful trim of block face, soak the block on ice water for a short time, return to holder, and use a brand new sharp edge to section.  Do NOT cut away what you have soaked, but over soaking will cause the decalcified bone to swell out of the block face, not a good thing.  You should be able to get good sections mounted on plus charge slides, but dry them flat at 37C to 40C overnight but several days is even better.  Do NOT go to a hot 60C oven.  Over drying can cause precious section lift off.   Good luckGayle Callis   HTL(ASCP)HT,MT, Bozeman MT