Hao, It might be possible that not all of the mountant has been removed. Though I am surprised that the staining is not better. It just might be one of those instances where the keratin epitopes are damaged during the 5 year storage. Possibly the phosphotungstic acid, often included in the PAP stains, might also be effecting the epitopes. Again try these variables on buccal or sputum smears and compare the results.
Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145 -----Original Message----- From: hao zhang [mailto:[EMAIL PROTECTED] Sent: Monday, 15 September 2008 12:22 PM To: Tony Henwood Subject: Re: [Histonet] antigen retrieval for 5 year pap smear slides Hi, Tony, René and all, I agree that pap smear is fixed by alcohol instead of formalin. Problem for me now is that when I do IHC for pan-keratin stain I only find small part cell stained (around 10%) even when I dont do antigen retrival by HIER. I know it is around 5 year old slides but I dont know why I can not get higher positive percentage because I used Hela cell as a control I got around 90% positive percentage. In addition, I find a lot of pink color material remained in the slide. I used 3% acid alcohol for destaining treatment and destaining effect get improved but finally I still get less positive percentage for pan-keratin. any suggest will be very appreciated. On Sun, Sep 14, 2008 at 5:26 PM, Tony Henwood <[EMAIL PROTECTED]> wrote: Do not bother to retrieve. Usually PAP smears are not fixed in formalin but are alcohol fixed. Alcohol is an excellent fixative for intermediate filaments as well as keratins. I would just de-coverslip the slides, rinse in buffer, block endogenous enzyme, add your diluted antibody (you will probably find that you may need to dilute your antibody further than for FFPE sections (maybe up to 1-100 times more)) and finally demonstrate the bound antibody. For titreing prepare some buccal smears, fix in the same fixative used for the PAP smears (95% ethanol or whatever commercial Cyto spray fix that is used), and work up the titreing as usual. No antigen retrieval is required unless formalin cross-links have been formed. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145 -----Original Message----- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of hao zhang Sent: Sunday, 14 September 2008 1:12 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] antigen retrieval for 5 year pap smear slides Hi, everyone I want to do IHC from a 5 year old pap smear slides for pan-keratin stain. I tried many methods of antigen retrieval protocols (10 mM citric buffer, ph 6; 1 mM EDTA, Ph8; 10 mM Tris buffer, Ph 10; ) and heat at 95C for 40 minutes. I also used Triton x 100 (0.2%) pretreatment 10 minutes and then SDS (0.5% or 0.25%) treatment for 10 minutes. But all method did not work. Any replies will be very appreciated. -- Hao ZHANG, Ph.D Scientist _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ********************************************************************* This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. ********************************************************************** -- Hao ZHANG, Ph.D Scientist Diamics, Inc ********************************************************************* This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. ********************************************************************** _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet