[Histonet] antigen retrieval for 5 year pap smear slides

[Tony Henwood]

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:haozhang1 <@t> gmail.com] 
	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 <AnthonyH <@t> chw.edu.au> 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: histonet-bounces <@t> lists.utsouthwestern.edu
		[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of hao
		zhang
		Sent: Sunday, 14 September 2008 1:12 PM
		To: histonet <@t> 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
		
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	-- 
	Hao ZHANG, Ph.D
	
	Scientist 
	Diamics, Inc
	
	


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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.
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