[Histonet] IF staining on peritoneal macrophages

[Adam .]

The best way I know to get cells to stick is to first cytospin liquid with
some protein (BSA or serum) onto the filters to pre-wet them. After that's
done, cytospin your cells. I've gotten this to work with fairly rare FACS
sorted cells.

I've also found that the smaller volume the cells are in, the better your
cytospin. Ideally is 100 - 200 uL. For FACS sorted cells, this means you
want to sort into as small a volume as possible. For lavage cells, that may
mean spinning them down first, although for FACS cells, the cells are
fragile and don't like being centrifuged.

I've gotten cells to stick to the slides by circling the cells with a PAP
pen and then just putting fixative inside PAP while the slides are lying
horizontally.

Adam

On Thu, Feb 18, 2010 at 5:00 PM, Mauricio Avigdor
<bitesizellama <@t> gmail.com>wrote:

> 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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