[Histonet] Problems in processing rat paws

[Elizabeth Chlipala]

Catia

Its going to take about a week to decal those types of samples in formic acid and in addition to that the processing cycle needs to be considerably longer.  By rat paw, I'm assuming you are mean the front paw or is it the rear ankle that you need to process.  First of all the samples need to be fixed for at least 48 hours prior to decalcification.  We use 10% formic acid in distilled water, seems to work fine for us.  For ankle samples without the rear toes, it takes about a week to decal.  If the toes are attached in our hands it takes about two weeks, we change the decal about every third day or so and always on the day prior to processing.  Ankles are trimmed in half and processed, both halves in one cassette.  For ankle and toes, we trim one side of the joint off prior to processing.  For front paws since they are a bit thick I would trim off some of the wrist pad prior to processing.  Processing cycle is long.  1 hour in each station for ankles and 1 to 2 hours per station for ankles with toes.  We use three absolutes and three xylenes and 4 paraffins when processing.

70% - 1 hour
80% - 1 hour
95% - 1 hour
100% - 1 hour
100% - 1 hour
100% - 1 hour
Xylene - 1 hour
Xylene - 1 hour
Xylene - 1 hour
Paraffin - 1 hour
Paraffin - 1 hour
Paraffin - 1 hour

Good luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308-1592
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
www.premierlab.com

Ship to address:

1567 Skyway Drive, Unit E
Longmont, CO 80504

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Catia Lopes
Sent: Friday, November 18, 2011 10:05 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Problems in processing rat paws

Hello everyone!
I have a problem in processing rat paws to paraffin embedding.
I need to process and cut the whole paw (except fingers). So, first I need
to decalcify the paw and then process it to paraffin embedding.
My problem is that when I try to cut the paw I can not get a good section
because the tissue have a gum-like consistence (mushy).
Do you know where is my problem? Dehydration, clearing, embedding?
**
*Note*: I decalcify the paws in  formic acid and aluminum chloride for 24h;
Then I wash samples in phosphate buffer saline for 5 hours. After that I
did the following manual processing (applying vacuum): 2x30min in 70%
ethanol, 2x30min in 80% ethanol, 2x30min in 96% ethanol, 2x30min in
absolute ethanol, 3x15 min in Xilene, 2x15min in paraffin and a final
incubation of 30min in paraffin.
Then, I proceed to paraffin ambedding and try to cut sections of 5um of
thickness.

If anyone can help me I will be grateful.
Thanks in advance,
Cátia
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