----- Original Message ----- From: Christopher Pin <[email protected]> Date: Thursday, January 28, 2010 9:22 Subject: Re: Stains for Macrophages for Laser Capture Microdissection purpose To: Martin Sandig <[email protected]>, John Kiernan <[email protected]>, Paul Walton <[email protected]>
> Hi all, > > Thanks for including me in these discussions. I am no expert on macrophage > histology but I know something about laser capture microdissection. The key > to this method for RNA isolation is not so much in the timing but rather in > the solutions complete lack of RNAse. We have worked with pancreatic tissue > and been able to dissect of cells for RNA isolation. Likely one approach > would be to do H&E for identification. Before you went to the trouble of > doing LCM, you could take some unimportant tissue, scrape the tissue off the > slide after fixation and then isolate RNA just to verify the integrity of the > RNA following staining. > > The other thing that you can do is fix the slides as if you are doing in situ > hybridization before you do any histology. > > I know this isn’t that helpful but from my experience it is not the time but > rather the quality of the solutions that makes all the difference. > > Chris > > > On 1/28/10 9:01 AM, "Martin Sandig" <[email protected]> wrote: > > Hi: > I do not do laser capture at all. > For identification purposes, perhaps it would be possible to load the > macrophages with an injected dye (they eat that stuff depending on the tissue > under investigation) and then capture them. In atherosclerotic lesions they > are loaded with lipids (foam cells) and perhaps with an excellent microscope > they may be visible before capture. > Chris Pin ([email protected]) may have some experience with laser micro-dissection. > Cheers, > Martin > > > Martin Sandig, PhD > Associate Professor > Associate Chair, Undergraduate Affairs > Department of Anatomy and Cell Biology > Schulich School of Medicine and Dentistry > University of Western Ontario > Dental Sciences Building, Room 00075 > Phone: 519 661 2111 86815 > Fax: 519 850 25662 > http://www.uwo.ca/anatomy/department/sandigm/msandig.html > > > > >>> Paul Walton <[email protected]> 28/01/2010 8:24 am >>> > John, > I have forwarded this message on to Martin, who does laser capture. Alas, I > am the microinjection fellow. > Paul > > Begin forwarded message: > > From: John Kiernan <[email protected]> > Date: January 27, 2010 10:41:15 PM EST (CA) > To: delphine eberle <[email protected]> > Cc: [email protected], [email protected], [email protected] > Subject: Re: Stains for Macrophages for Laser Capture Microdissection purpose > > How about phase contrast optics and identifying the cells by their shapes? > Non-specific addition of colour to a section of unfixed tissue probably will > not show the cells any more clearly. I have taken the liberty of including a > colleague, Dr Paul Walton, in this reply. He does laser capture > microdissection and may have something to suggest. > > John Kiernan > Anatomy, UWO > London, Canada > = = = > ----- Original Message ----- > From: delphine eberle <[email protected]> > Date: Wednesday, January 27, 2010 16:27 > Subject: Stains for Macrophages for Laser Capture Microdissection purpose > To: [email protected], [email protected] > Cc: [email protected] > > Hi, > > > > I have another question following Macrophage staining. > > I am setting up a Laser Capture Microdissection protocol to dissect > > macrophages from atherosclerosis lesions (non fixed frozen sections) and > > extract RNA for gene expression purpose. > > I am looking at a quick staining (less than 30min otherwise RNA is > > degradated) for macrophages in that context? Any suggestions? > > > > Thanks a lot, > > Delphine > > > > Delphine Eberlé PhD > > UCSF Department of Vascular Surgery > > [email protected] <java_script:main.compose('new', > > '[email protected]')> > > > > > From: [email protected] > > > To: [email protected] > > > Date: Wed, 27 Jan 2010 00:22:53 -0500 > > > Subject: Re: [Histonet] Histology Special Stains for Macrophages > > > CC: [email protected] > > > > > > None of the methods mentioned in the enquiry are stains for macrophages. > > > Research workers who never took Histology 101 often stain cells of the > > > monocyte/macrophage lineage immunohistochemically (IHC), using very > > > expensive primary antibodies and fairly expensive kits to amplify and > > > detect the binding sites. IHC is necessary if you must find every > > > macrophage, including a tissue's recent monocyte immigrants that haven't > > > yet done any work. Macrophages that have been busily eating blood are > > > full of brown granules that don't need any staining. > > > > > > Ordinary people recognize macrophages by their appearance in sections > > > stained with a well done H&E or with one of the many Romanowsky-Giemsa > > > blood stains. With the latter it's important to get the pH right - much > > > lower for sections of formaldehyde-fixed objects (pH4 to 5) than for > > > methanol-fixed films or smears (traditionally 6.8). It's all very well > > > explained in RD Lillie & GM Fullmer's "Histopathologic Technic..." (4th > > > ed 1976, pp.193-197; the 3rd edition, 1965, is OK too) and also in more > > > recent textbooks including those by Bancroft & Gamble, Geoffrey Brown, > > > Freida Carson and, of course > > > Yours truly. > > > > > > John Kiernan > > > Anatomy, UWO > > > London, Canada > > > http://publish.uwo.ca/~jkiernan/bookfind.htm > > > = = = > > > ----- Original Message ----- > > > From: "Willman, Sharon" <[email protected]> > > > Date: Tuesday, January 26, 2010 12:12 > > > Subject: [Histonet] Histology Special Stains for Macrophages > > > To: "[email protected]" > > > <[email protected]> > > > > > > > Hi, > > > > We are needing to do a special stain for macrophages. What > > > > is the most common stain for that? Does anyone do a Sudan > > > > Black, Alcian Blue or Van Gieson for macrophages? Any > > > > information would be appreciated. > > > > Thanks in advance. > > > > Sharon > > > > > > > > > > > > ________________________________ > > > > This message (including any attachments) may contain > > > > confidential, proprietary, privileged and/or private > > > > information. The information is intended to be for the use of > > > > the individual or entity designated above. If you are not the > > > > intended recipient of this message, please notify the sender > > > > immediately, and delete the message and any attachments. Any > > > > disclosure, reproduction, distribution or other use of this > > > > message or any attachments by an individual or entity other than > > > > the intended recipient is prohibited. > > > > _______________________________________________ > > > > Histonet mailing list > > > > [email protected] > > > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > > _______________________________________________ > > > Histonet mailing list > > > [email protected] > > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > > > Vous cherchez l'intégrale des clips de Michael Jackson ? Bing ! Trouvez ! > > <http://www.bing.com/videos/search?q=Michael+Jackson&FORM=MVDE6> > > Paul Albert Walton, Ph.D. > Department of Anatomy and Cell Biology > #474 Medical Sciences Building > The University of Western Ontario > London, Ontario, CANADA N6A 5C1 > (519) 661-2111 x86825 > fax (519) 661-3936 > ------------------------- > > > > > > > > Christopher Pin, Ph.D. > Associate Professor > Depts. Of Paediatrics and Physiology & > Pharmacology > Schulich School of Medicine and Dentistry > University of Western Ontario > Scientist, Children's Health Research Institute _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
