Marina,

I would suggest the following approach;

1) Let the marrow smears fully air-dry and stabilize at room temperature for
20 -30 minutes prior to fixation in methanol ( use a fan if you have to).
2) Only use fresh pure methanol for each batch of slides and allow at least
10 mL of methanol for each slide. Increase the fixation time to 15 - 30
minutes to help remove fat.
3) After fixation, allow the slides to air dry, then store at room
temperature in a dessicator until staining.
We have successfully stained smears treated this way and stored for up to 3
months.
4) Re-hydrate the dry smears in acetate buffer pH 6.8 for 30 minutes @37C.
5) Stain in May-Grunwald, diluted 1:5 with pH 6.8 buffer for 10 -15 minutes.
6) Rinse in pH 6.8 buffer.
7) Stain in Giemsa, diluted 1:5 with pH 6.8 buffer for 30 minutes.
8) wash in pH 6.8 buffer for 2 -5 minutes until the normochromatic RBC
appear clear pink.
9) If the normal RBC'c persist in having a blueish tone then consider
lowering the PH of the final wash buffer system to pH 6.5 or lower(as low as
pH 5.8 may be necessary).
10) Drain and air dry.

Regards,

Bryan

----- Original Message -----
From: "marina goumenou" 
To: 
Sent: Thursday, October 03, 2002 4:30 PM
Subject: May Grunwald - Giemsa problems!


> Hi to all Histonetters
>
> I work with May Grunwald - Giemsa for about 2 years staining bone marrow
> smears from rats (I count micronucleus in polychromatic RBC) but the
results
> are not constantly well. So, I still search to find what is going whrong.
> Some of the artifacts I have noticed are:
> Very bright RBC, green RBC, no diferentiation between normochromatic and
> polychromatic RBC, breaked RBC, purple in the center of normochromatic RBC
> etc. I thought many different possible reasons for all these problems and
I
> try to stabilize the parameters of the protocol I use but I am not sure
> about my thoughts:
> I use DI water instead of buffer, is this sufficient? (I am not sure that
my
> water is constantly well)
> I filter Giemsa before use but what kind of filter is appropriate (pore
> size, material etc)?
> I use stock solutions (botlles of 2.5L) and some times they are not in use
> for weeks after I open them. Is this a possible problem?
> How long can I store my smears after fixation (5min in methanol) without
> staining them?
> Is it possible having burst RBC due to FBS osmolarity and/or water in
> methanol during fixation?
>
> I really need any help, any thought, any technical information!
> Thanking you in advance
> Goumenou Marina
> Athens-Geece
>
>
>
>
>
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