[Histonet] Fwd: Re: Stains for Macrophages for Laser Capture
Microdissection purpose
John Kiernan
jkiernan <@t> uwo.ca
Thu Jan 28 09:56:55 CST 2010
----- Original Message -----
From: Christopher Pin <cpin <@t> uwo.ca>
Date: Thursday, January 28, 2010 9:22
Subject: Re: Stains for Macrophages for Laser Capture Microdissection purpose
To: Martin Sandig <Martin.Sandig <@t> schulich.uwo.ca>, John Kiernan <jkiernan <@t> uwo.ca>, Paul Walton <pwalton <@t> uwo.ca>
> 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" <Martin.Sandig <@t> schulich.uwo.ca> 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 (cpin <@t> uwo.ca) 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 <pwalton <@t> uwo.ca> 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 <jkiernan <@t> uwo.ca>
> Date: January 27, 2010 10:41:15 PM EST (CA)
> To: delphine eberle <eberledelphine <@t> hotmail.com>
> Cc: sharon.willman <@t> bms.com, histonet <@t> lists.utsouthwestern.edu, pwalton <@t> uwo.ca
> 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 <eberledelphine <@t> hotmail.com>
> Date: Wednesday, January 27, 2010 16:27
> Subject: Stains for Macrophages for Laser Capture Microdissection purpose
> To: jkiernan <@t> uwo.ca, sharon.willman <@t> bms.com
> Cc: histonet <@t> lists.utsouthwestern.edu
> > 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
> > eberledelphine <@t> hotmail.com <java_script:main.compose('new', 't=eberledelphine <@t> hotmail.com')>
> >
> > > From: jkiernan <@t> uwo.ca
> > > To: sharon.willman <@t> bms.com
> > > Date: Wed, 27 Jan 2010 00:22:53 -0500
> > > Subject: Re: [Histonet] Histology Special Stains for Macrophages
> > > CC: histonet <@t> lists.utsouthwestern.edu
> > >
> > > 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" <sharon.willman <@t> bms.com>
> > > Date: Tuesday, January 26, 2010 12:12
> > > Subject: [Histonet] Histology Special Stains for Macrophages
> > > To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
> > >
> > > > 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
> > > >
> > > >
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>
> 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
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