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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Catia Lopes Sent: Friday, November 18, 2011 10:05 AM To: histonet@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
