|From:||Gayle Callis <email@example.com>|
One alcohol containing fixatives has NOT destroyed our murine CD4 or CD8
epitopes, but the fixative does contain acetone. Mixture is 75%
acetone/25% absolute ethanol 5 min at RT on overnight air dried frozen
sections. This recipe was passed on to me by Barb Wright at Genetech. The
joy of using it, improved morphology!
We have our biotinlylated CD4 antibody (1 mg/ml) diluted out 1:20,000
incubated RT for 30 min after a Glucose oxidase/glucose/sodium azide endog
perox block, SA-HRP for 20 min, and DAKO DAB+/enhancer. This is not a kit
method and we use a Normal serum block, inhouse preparation, with biotin
conj primary diluted in same block. Our CD8 is also more dilute than we
had previously used with this protocol.
I would not want to fix my sections or cells preps with just alcohol, but
this AA fix works for us.
AS in all things, a fixative study may be in order to know WHICH one gives
optimal results at what times and temperatures, and the acetone/alcohol
mixture may NOT be optimal for other peoples CD markers.
>Date: Wed, 29 Nov 2000 09:21:25 +0100
>From: "C.M. vander Loos" <firstname.lastname@example.org>
>Subject: Re: CD4
>To: "Dr. Ian Montgomery." <email@example.com>
>> Following on from Nima's posting, I'll be staining cultured cells
>>for CD1 and CD4 using Dako antibodies. Any suggestions for the preferred
>>fixative would be welcome.
>> Dr. Ian Montgomery,
>> West Medical Building,
>> University of Glasgow,
>> G12 8QQ.
>> Tel: 0141 339 8855. Extn:6602.
>> Fax: 0141 330 2923
>> e-mail: firstname.lastname@example.org
>Following our experiences with the immunostaining of intact cultured cells,
>I think you should test both acetone (destroying the fatty membrane
>structures) and 4% paraformaldehyde in PBS (2-5 min, RT). When using PFA
>the addition of 0.1% saponin to reagents and washing buffers can be skipped
>since this seems only useful for staining cytoplasmic antigens. Forget
>about alcohol containing fixatives, as these will destroy the CD1/CD4
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
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