Dear Colleagues Please remove my husband, Charles E Brown Jr. from you listings. My husband passed away on 1/27/11. Respectfully Mrs. Charles E Brown, Jr.
-----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of gayle callis Sent: Wednesday, February 09, 2011 4:42 PM To: 'Histonet' Subject: [Histonet] RE: Tape Transfer You Wrote (two messages) Dear Nele, I hear of these problems very often. It would be worth your time looking for any information on fresh frozen bone sections by Dr. Dodds, there should be a number of papers he has published on good quality bone sectioning without the use of the slow, expensive and troublesome tape method. Alan Bright www.brightinstruments.com Sent from my BlackBerryR wireless device -----Original Message----- From: "Nele Degryse" <Nele.Degryse <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> UGent.be> Sender: histonet-bounces <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> lists.utsouthwestern.edu Date: Wed, 09 Feb 2011 09:30:44 To: <histonet <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> lists.utsouthwestern.edu> Subject: [Histonet] Tape transfer Hello, I'm trying to cut undecalcified bone (mouse knees) by using the tape transfer method. The problem is that the area that I'm interested in (the joint with patella) is not transferred from the tape on my slide. I've used slides with different coating (also those specially made for bone) and tried different thickness. None of that seems to make a difference. Another problem is that my hand roller started to stick, so I cleaned it with 100% ethanol (as recommended) and now it sticks even more... Can anybody give me some advice? **************************************************************************** *********************************** First, I totally disagree that using the "slow, expensive and troublesome" tape method is a problem. I took Dodd's workshop and then attempted cut bone frozen sections in our lab using his methods which proved to be a nightmare, taking hours to get one decent section IF we were lucky. We never "got lucky" in obtaining a an intact bone section that was free from folding and totally disrupted morphology. If any method is troublesome, it is the Dodd's method plus the expense of wasting time and nothing to show for it. The only thing I have used with any success was snap freezing bone the way he taught us, but that has gone by the wayside due to the toxic hexane fumes. His way was a total failure in our laboratory and we bought the Cryojane to get decent bone sections. Cryojane requires practice, a bit of patience, fine tuning the little details for successful use and it is not as slow as people think. There are many labs who use it successfully including ours. First of all, Nele did not provide any details on how the Cryojane was being used other than the different polymer coated slides. So questions follow: What was the cryostat chamber temperature? Is this the same temperature of the sample, knife and UV light source? If the cryostat is older model, have you actually checked the temperatures? Is the bone fixed in formalin before snap freezing? And if the bone was fixed, was it cryoprotected with at least 20 to 30% sucrose (this takes a long time for NBF fixed bone)? What thicknesses did you try? Thinner is often better than thicker. Is the d profile tungsten carbide knife perfectly sharp when cutting the frozen bone sections? This means a new edge for sectioning, and very frequent reconditioning of the knife (DDK or Dorn and Hart sharpening services). Hopefully you have two knives. Have you tried multiple UV flashes? Two to three flashes usually help keep bone on the slide. Do you remove the tape INSIDE the cold cryostat environment, and at an angle from one corner of slide across the section, slowly and gently. Have you tried orienting the bone differently? Sometimes this helps, particularly when removing the tape from the bone. Is your mouse leg left articulated so you section through the bent knee joint (into patella)? It sounds as though you have a temperature problem IF things are sticking. When washing the roller, are you putting a lot of alcohol on the roller, or simply wiping the roller with the alcohol. Is the roller at the exact cutting temperature of the sample before rolling the COLD tape onto the block face? More details will help since we can't see a real time session of what you are doing. Gayle M. Callis HTL/HT/MT(ASCP) _______________________________________________ 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