[Histonet] Re: Stains for Macrophages for Laser CaptureMicrodissection purpose

[Percival Karen]

Delphine,
 
I think John is right about identifying the cells by shape.  I do LCM, and I perform a rapid hematoxylin stain just for orientation purposes.  The total time from fixation to air drying is 7 minutes.  Any more time than that, and the RNA is too degraded to get good results from downstream applications.  You could substitute toluidine blue for the hematoxylin in this case.  Are you really looking for a (no greater than) 30 minute (aqueous) stain?  Seems way too long for good quality and quantity RNA.  I would be interested in hearing what you usually do for staining for LCM purposes and what your RNA results have been.
 
Karen
 
Karen Percival, BS, HT
Research Scientist II
Pfizer Research DSRD
1 Burtt Road
G3025
Andover, MA 01810
888-577-1500 x 4058
kpercival @wyeth.com

>>> John Kiernan <jkiernan <@t> uwo.ca> 1/27/2010 10:41 PM >>>

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 
> 
> > 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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