Abcam Ad

Author Topic: A good Counter stain  (Read 8627 times)

0 Members and 1 Guest are viewing this topic.

Offline ahiscox

  • HighTech
  • ***
  • Posts: 15
A good Counter stain
« on: November 20, 2007, 10:14:15 AM »
I am in the process of developing a protocol for IHC using HRP-conjugates, and was wondering what kind of counterstain works well.

I have two protocols written (to be tested next month):

1.  go through the staining process (primary, secondary, substrate), then counterstain with copper sulfate-methyl green.

or

2.  Before beginning the staining process, do a quick H&E stain, then start the IHC stain.


Any comments about a good counter staining method would be greatly appreciated.

A good Counter stain
« on: November 20, 2007, 10:14:15 AM »

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
Re: A good Counter stain
« Reply #1 on: November 20, 2007, 06:13:31 PM »
It depends on what color product you plan to use. If you use DAB-brown, the best counterstain will be hematoxylin or methyl green. If you use DAB-blue, the best will be nuclear fast red.

Do not stain section with H&E if you plan to stain the it with IHC. Otherwise you will not see IHC staining well. Always do IHC First and then counterstain.

Offline ahiscox

  • HighTech
  • ***
  • Posts: 15
Re: A good Counter stain
« Reply #2 on: November 21, 2007, 09:46:49 AM »
Thanks. 

I am using DAB-brown, so I guess I'll just try both counterstains and see which one looks best for my application.

Thanks for the advice about doing IHC before H&E.  It's strange how everything seems like common sense AFTER you're told which way is better.

Offline excalibur

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 1109
    • http://www.excaliburpathology.com
Re: A good Counter stain
« Reply #3 on: November 21, 2007, 11:58:01 AM »
This is an excellant reference for counterstains for IHC from Vector.

http://www.vectorlabs.com/Protocols/Counterstains/cstaincomp.pdf
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline gussy

  • SuperTech
  • ****
  • Posts: 38
Re: A good Counter stain
« Reply #4 on: November 27, 2007, 08:10:31 PM »
Dear Forum I also have question about counter staining, I usually counter staining with Meyer Hematoxillin but still can not find good result sometimes it is too pale (only slighly blue) or too dark (makes IHC stining become dark brown) I usually counterstaining for 5 minutes in Hematoxilin and then wash in running tap water for 10 minutes, what is the best way for counterstaining ( I read only 30 second in IHC protocol) di I also need to wash in running tap water? thanks

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
Re: A good Counter stain
« Reply #5 on: November 27, 2007, 10:34:20 PM »
First of all, 5 minutes for "IHC Counterstain" is TOO long. I normal stain only 10 seconds for fresh solution. After re-use several times, I stain 20-30 seconds but never go over 1 minutes.

Rinsing in tap water depends on color change of nuclei. You need to check slides microscopically for the best stopping point. It may range from 1 minutes to 5 minutes or longer.

Anyway, there is no standard rule as the hemo reagent you bought from one company may be quite different from another. You may need to do a test and find the best condition such as staining time, dilution, etc.

Hope this help.

Dear Forum I also have question about counter staining, I usually counter staining with Meyer Hematoxillin but still can not find good result sometimes it is too pale (only slighly blue) or too dark (makes IHC stining become dark brown) I usually counterstaining for 5 minutes in Hematoxilin and then wash in running tap water for 10 minutes, what is the best way for counterstaining ( I read only 30 second in IHC protocol) di I also need to wash in running tap water? thanks

Offline gussy

  • SuperTech
  • ****
  • Posts: 38
Re: A good Counter stain
« Reply #6 on: November 28, 2007, 07:56:40 PM »
Oke,thank you very much for your suggestion, I will make the test as you say

Re: A good Counter stain
« Reply #6 on: November 28, 2007, 07:56:40 PM »