[Histonet] IF staining on peritoneal macrophages

[Andrea T. Hooper]

After cytospinning I always dry my slides thoroughly before moving on to a IHC or IF step. I also use Superfrost Plus slides or silane coated slides to increase adherance. I would do that then try various fixatives.
 
You can make a cell pellet as you suggest. Decide ahead of time if you want to fix before embedding (ie: is your antigen preserved in PFA). If so, fix in suspension then spin and resuspend in a small volume of 2% agarose (or Histogel). Then you can embed in OCT for frozen sectioning or wax for FFPE.
 
- Andrea Hooper


--- On Thu, 2/18/10, Mauricio Avigdor <bitesizellama <@t> gmail.com> wrote:


From: Mauricio Avigdor <bitesizellama <@t> gmail.com>
Subject: [Histonet] IF staining on peritoneal macrophages
To: histonet <@t> lists.utsouthwestern.edu
Date: Thursday, February 18, 2010, 11:00 PM


Greetings all,

I am trying to do immunofluorescence on peritoneal macrophages. I am having
a couple of issues that I was hoping one of you could help me resolve.

Firstly, I am having uneven results with the Cytospin. Cells tend to get
washed off the slides during rinses. Does anyone have tips on how to make
cells stick a little better to Cytoslides? Secondly, I have not yet found a
satisfactory method for fixation. In order to prevent loss of cells when
dipping the slides into fixatives, I am having to air dry the slides before
I can do anything to them. I tried the Shandon Collection Fluid (ethanol,
isopropanol, carbowax) with great success, but it killed the fluorescence.

I am leaning towards spinning the lavage fluid until I get a pellet and then
resuspending in PBS with a little BSA added. I hope this makes cells stick
well enough that I can put the slides in formalin or acetone.

Any thoughts are appreciated!
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