I'm afraid too that immunostaining for CD's is extremely difficult if not impossible, we make a lot of experiments in determining optimal fixation procedure for immunodetection of CD's and other antigens in human cytological specimens and we found as some other investigators that drying of the cells before fixation is detrimental for many antigens especially for surface antigens! it is true that you can detect some robust antigens like CK even in smears previously stained with Giemsa, but this is exception.
Maybe you should try with microwave pretreatment or you can ask clinicians to use spray fixative for fixing the smear or to prepare you the cells suspension.
hope this helps
From: Chris van der Loos [mailto:firstname.lastname@example.org]
Sent: Tuesday, April 22, 2003 9:34 AM
Subject: RE: ask for a protocol for CD3 or CD79a on previously stained
Dear Ul Soo,
As those antigens are probably heavily damaged by staining and storing the
stained slides at room temperature (??), I am afraid CD1a, CD79a and CD3
IHC staining is going to be extremely difficult, or perhaps impossible. The
only change you have is trying the tyramide amplification IHC procedure. A
kit system by Perkin & Elmer (NEL-700) provides the highest amplification
possibilities for IHC right now. Lots of success.
Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Center
Amsterdam - The Netherlands
Ul Soo wrote:
>Date: 21 Apr 2003 12:30:23 -0600
>From: Ul Soo <email@example.com>
>Subject: ask for a protocol for CD3 or CD79a on previously stained slides....
>I'm asking for a protocol for applying CD1a, CD3 or CD79a on previously
>stained slides, especially by Romanoswsky type stains.
>I'm doing diagnostic cytology in dogs and cats, and I would like to to set up
>a protocol for previously stained slides by Wright, Giemsa, or May Grunwald
>Giemsa stains, because, there are increasing tumor cases where we can only
>obtain a few stained cytologic smears and no mass excised for confirmation!
>Pet owners avoid surgery frequently for finacial constrains , poor prognosis
>or lack of information for successful treatment.
>BUt NO tentative diagnosis without histologic or immunohistochemical
>confirmation bear any meaning in clinical oncology!! In fact, I have
>to apply some antigens on Wright stained slides and got some successful
>results, but as for CD antigens such as CD1a, 3, or 79a I have had no idea! I
>have used antigen retrieval system using citrate buffer,pH6.0 after smear
>trasfer using Mount Quick to adhesive slides, but the results went
>unsuccesful. Any help in any form would be appreciated. If I have ever tried
>awful things with CD antigens so far, please let me hear No way, or if anyone
>with experiances immunoCYTOchemistry on Wright stained slides using CD
>antigens, give me tips for the procedures. Any cautions or advice, or
>information based on literature would be great help to me.
>A protocol for applying CD antigens on Wright stained cytologic smears!
>Thank you in advance.
>Ul Soo, Choi
>DVM, graduate student
>Dept of clinical pathology, Veterinary college
>Seoul Nat'l Univ, South Korea