[Histonet] RE: immunohistochemistry help please

[Tony Henwood (SCHN)]

Rebecca,

Try making sure the sectioned slides have been adequately dried (up to 65oC for at least 30 minutes) before de-waxing.
I believe that much of the de-waxing of sections occurs during the slide drying phase and this is not given the importance it deserves.
You could also try heated detergent dewaxing:
Henwood A (2012) "The application of heated detergent dewaxing and rehydration to immunohistochemistry" Biotechnic & Histochemistry 87(1):46-50.
Henwood, A. F., Prasad, L., & Bourke, V. M. (2013). "The application of heated detergent dewaxing and rehydration to techniques for the demonstration of fungi: a comparison to routine xylene-alcohol dewaxing" J Histotechnol 36(2):45-50.


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
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, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tarantelli, Rebecca Anne
Sent: Saturday, 3 August 2013 12:30 AM
To: Sebree Linda A; histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: immunohistochemistry help please

Thanks Linda, 

This is going to be a very dumb question (may I remind you this is my very first IHC experience) how do I know the melting point of my paraffin? It was not a new bottle of xylene, and the tissues are all from the same block cut into sequential slides.... 


Rebecca 


-----Original Message-----
From: Sebree Linda A [mailto:LSebree <@t> uwhealth.org] 
Sent: Thursday, August 01, 2013 3:50 PM
To: Tarantelli, Rebecca Anne; histonet <@t> lists.utsouthwestern.edu
Subject: RE: immunohistochemistry help please

OK, so I would be very suspect of your xylene.  Has anything changed, i.e. vendor?  It sounds like it's not dissolving your paraffin.  Are you using the same paraffin you were when you were successful?  Do you dry/melt your slides in an oven prior to depar?  If not, you could try that at a  temperature just above the melting point of your paraffin for 10 - 30 minutes.

Linda A. Sebree
University of Wisconsin Hospital & Clinics IHC/ISH Laboratory
600 Highland Ave. 
Madison, WI 53792
(608)265-6596
FAX: (608)262-7174 


-----Original Message-----
From: Tarantelli, Rebecca Anne [mailto:rat37 <@t> pitt.edu]
Sent: Thursday, August 01, 2013 2:40 PM
To: Sebree Linda A; histonet <@t> lists.utsouthwestern.edu
Subject: RE: immunohistochemistry help please

Hi Linda, 

I'm sorry - I should have been a little more clear. Once I realized it isn't working, I did stop using the antibody. I have been trying just deparaffinization, and then counter staining with hematoxylin and skipping everything in between. The tissue isn't taking up the hematoxylin. With your experience, would you have any suggestions as to what part of the deparaffinization stage would cause the hematoxylin not to stain? I use: Xylene 3 minutes x 3, 100% ethanol 3 minutes x 2, 90 % ethanol 3 minutes x 1, 75% ethanol 3 minutes x1, distilled water 2 minutes x 1, then Dako wash buffer 3 minutes x 1. Next, I have been just going right to 3 minutes in my hematoxylin. 

I'm sorry if these are beginner questions, this is my first attempt at IHC, and it worked so well for a week.... and now nothing. 

Thank you!

Rebecca Tarantelli


-----Original Message-----
From: Sebree Linda A [mailto:LSebree <@t> uwhealth.org]
Sent: Thursday, August 01, 2013 3:36 PM
To: Tarantelli, Rebecca Anne; histonet <@t> lists.utsouthwestern.edu
Subject: RE: immunohistochemistry help please

To test your deparaffinization Rebecca, I'd try staining a slide with something cheaper and quicker, like just your counterstain to see if that's working.  Then go from there.

Linda A. Sebree
University of Wisconsin Hospital & Clinics IHC/ISH Laboratory
600 Highland Ave. 
Madison, WI 53792
(608)265-6596
FAX: (608)262-7174 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tarantelli, Rebecca Anne
Sent: Thursday, August 01, 2013 2:20 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] immunohistochemistry help please

Hi all,

I am new to this forum, but a friend recommended I send my issue out to you for help. Four weeks ago I completed a new protocol for Immunohistochemistry (IHC) using protinase K epitope retrieval, and monoclonal antibody 3F6. It worked perfectly. Three days later, after 13 beautiful stains, it just stopped working. The slides will not even take up the counter stain - Hematoxylin. I have exchanged my xylene, and tried again, and it still is not working. I am guessing since my counter stain isn't working, this is an issue in the deparaffinization stage, but I'm just not sure.

Please let me know if you have any suggestions!

Thank you
Rebecca

Rebecca Tarantelli

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