Hi Tony: Thanks a lot for the correction! I am in research institute, quite a gap...
2008-12-17 tf 发件人: Tony Henwood 发送时间: 2008-12-17 06:07:48 收件人: ti...@foxmail.com; Pat Flannery; histo...@lists.utsouthwestern.ed 抄送: 主题: RE: (reply) silly questions.---PFA tf, Answers as follows: Seems obvious that the 4% formaldehyde (made from PFA) was incorrectly made. What they did, I have no idea nor did I have the time to show them how to make it up. My advice: "make it from concentrated 38% formaldehyde (formalin)". You wrote: "I do think most biomedical labs currently are using PFA to prepare the fixatives!" - I do not think so. A sweeping statement if ever I have heard one. This s the type of misinformation that we have to deal with. My experience (over 30 years) indicates that all pathology labs I have worked in as well as those I have visited, as well as several Pathology Colleges (eg CAP and RCPA) surveys indicate that MOST (if not all) histopathology labs prepare their 10% formalin fixative from concentrated formalin not polyformaldehyde as you have incorrectly stated. Be careful of misinformation. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -----Original Message----- From: tf [mailto:ti...@foxmail.com] Sent: Friday, 12 December 2008 7:35 PM To: Tony Henwood; Pat Flannery; histo...@lists.utsouthwestern.ed Subject: (reply) silly questions.---PFA "I looked at the sections and the cell shrinkage (and prominent spaces between cells and connective tissue) indicated that most of the "fixation" seemed to have occured in the processing ethanols. I asked him for some of the fixative he used, tested the formaldehyde concentration and found it to be less than 0.5%!!" Tony: Do you think this is because of inproper preparation of PFA in his lab, or the common problem in all researchers using PFA? I do think most biomedical labs currently are using PFA to prepare the fixatives! So, anyone has the idea on a correction preparation procedure of 4% PFA? I noticed some of you dissolve PFA powder in NaOH-conditioned alkaline water, then add concentrated PB solution. We here dissolve PFA in concentrated PB solution directly (heat & stir for 2-3 hours), then adjust pH to 7.4. We dont have big problem in tissue quaility....except when one want to cut the brain in a cryostat rather sliding microtome. Many times the brain sections from the cryostat have "cheese" like holes/cavities, which almost never appear on sliding microtome-prepared sections. 2008-12-12 tf 发件人: Tony Henwood 发送时间: 2008-12-12 06:18:47 收件人: Pat Flannery; histonet@lists.utsouthwestern.edu 抄送: 主题: RE: [Histonet] Silly Question? Pat, I agree with you. In a routine diagnostic histopathology laboratory, it makes little difference what you use. Around the world for over 100 years most labs use 10% neutral buffered formalin made from concentrated 38%(or there abouts) formalin (or formaldehyde). Researchers, though, are a different kettle of fish. They will tend to hang on to misinformed, "mystical" methods believing they are being scientific. Funny, you would think that they, as a group, would be the ones pushing the boundaries and critically assessing each step of their research, ensuring that they understand what and why they are doing it. (Disclaimer - not all researchers are like this, thank heavens!!) Using a formaldehyde solution made from polyformaldehyde can cause problems. One researcher used it and wondered why their morphology was sub-optimal and their p53 immunohistochemistry was negative. He assured me that he had fixed small samples of tissue for 6 hours in 4% formaldehyde and then processed them using ethanol, xylene and wax. I looked at the sections and the cell shrinkage (and prominent spaces between cells and connective tissue) indicated that most of the "fixation" seemed to have occured in the processing ethanols. I asked him for some of the fixative he used, tested the formaldehyde concentration and found it to be less than 0.5%!! This also explains the loss of p53 staining. I gave him some of our routine 10% phosphate buffered fomalin, asked him to fix overnight, and try agin. Low and behold problem solved. How's that for a Friday Flamming!!! Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Pat Flannery Sent: Friday, 12 December 2008 3:59 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Silly Question? Please humor me on this if it's obvious (to everyone but me): why do we use paraformaldehyde (which is so inconvenient to make up) rather than buffered formalin or just diluted formaldehyde itself? It seems that around here, some folks prefer paraformaldehyde (either 2% or 4%) and others use formalin, while some others stick to diluted formaldehyde (I see all 4 on labels for specimens submitted for histology). Is it mostly a matter of personal preference or where you were trained (i.e. force of habit) or is there a valid reason to use each solution (basically the same chemical once in solution, merely buffered or not)? The only answer I've gotten when I've asked is, "That's what we always use." Thanks. -Pat Flannery (not a "real" histologist - I just play one in the lab) _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ********************************************************************* This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. 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