Author Topic: adequate counterstaining for DAB  (Read 17662 times)

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Offline Simone

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adequate counterstaining for DAB
« on: January 20, 2006, 10:45:03 AM »
I try to enumerate DAB-stained CD4- and CD8-T-lymphocytes in 10 m
cryostat-sections of porcine ileum.
I counterstained the sections with Mayer`s hematoxylin (10% aqueous
solution).

The counterstaining was performed as follows:
    1.    Hematoxylin for 15 sec
    2.    short dip into ethyl alcohol/ 0,1% HCl (regressive step)
    3.    5 min in tap water (for blueing)
    4.    One dip into double-distilled water
    5.    in each case 5 minutes:

                    *    50% ethyl alcohol
                    *    70% ethyl alcohol
                    *    99% ethyl alcohol
                    *    isopropyl alcohol

    6.    20 min in xylene
    7.    Mounting with Entellan (permanent mounting medium,non-aqueos)

Unfortunately, the contrast between DAB and the hematoxylin-counterstaining is too weak because the DAB-stained cells appear to be brown and hematoxylin produces a dark violet.
With this result it is not possible to enumerate the positive stained T-cells. With prolonged colouring in hematoxylin I get nicely distinguishable nuclei but the contrast to DAB gets even poorer.
Does anyone have an idea how to change the hematoxylin-protocol to achieve a better contrast? It would be nice if there is a way to turn hematoxylin from dark violet into a clear blue that I already observed in paraffin sections.
I`ve already tried forced blueing in ammonia water, without any mprovement.
Please tell me about your experiences with hematoxylin-counterstaining and DAB.
Or is it the best solution to try another dye like methyl green??

Thanks for your help,
Simone from Germany

adequate counterstaining for DAB
« on: January 20, 2006, 10:45:03 AM »

Offline excalibur

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adequate counterstaining for DAB
« Reply #1 on: January 20, 2006, 12:50:56 PM »
Mayer's does tend to give muddy staining. Try Gill II or III hemat.

And since you are using DAB,  try Nuclear Fast Red. It stains nuclei red.
Paula K. Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
8901 S Santa Fe, Suite G
Oklahoma City, OK 73139
405-759-3959
www.excaliburpathology.com

Offline ImmunoNYC

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adequate counterstaining for DAB
« Reply #2 on: January 20, 2006, 02:29:00 PM »
I use Mayer's everyday for my DAB counterstain. In my experience, red nuclei does not give enough contrast especially when quantitating.

Mayer's is a PROGRESSIVE stain not regressive.  You should not need to differentiate in acid alcohol.

For counterstain I do the following:

- After DAB I do several dH20 rinses.
- 5 to 15 seconds up to 1 minute (depending on how dark you want the stain) in Mayer's hematoxylin
- several washes in dH20 or tap water
- blue in Blueing reagent or ammonia water (this is crucial for BLUE not VIOLET color)
- dehydrate in graded alcohols and clear in xylene.

Gorgeous, crisp, every time.

I use Mayer's hematoxylin from DAKO (Lillie's modification) but I know Richard Allen's is a really good one too. I find Sigma's Mayer's to be terrible and oxidized. Where is yours from? Make sure it is NOT EXPIRED.

I think because you are staining T-cells which have a massive nuclei to cytoplasmic ratio it is crucial you keep your staining very LIGHT yet crisp.

Alternatively if desired you can use Harris' hematoxylin and differentiate in acid alcohol for even more increased crispness.
 
See my pics in my gallery for examples of the type of stain I get using Mayer's. I have pics of B cell and granulocyte staining with Mayer's counterstain which are comparable to your stain.

Offline richard03

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adequate counterstaining for DAB
« Reply #3 on: January 20, 2006, 10:30:13 PM »
My suggestion is to dilute your Mayer's 1:5 and then stain for 15 seconds or stain for ONLY 3 seconds using the concentrated Mayer's.

Bluing is also important. Bluing reagent from Richard Allan is a good one. You can also try one of these protocols (http://www.ihcworld.com/_protocols/histology/bluing.htm) and they are working as well.

I have recently switched to GILL from Vector Labs, it works well and no bluing is necessory.

Nuclear fast red (red/pink) is too close to brown. I have done this a few times for some double labeling projects and it is somewhat difficult to distinguish. However, methy green maybe an alternative.

Richard

Offline Simone

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adequate counterstaining for DAB
« Reply #4 on: January 21, 2006, 11:30:59 AM »
Thank you for your replies and helpful suggestions- new ideas are still welcome...
I use Mayer`s Hematoxylin from MERCK and I already made sure that it is not expired..so there has to be another reason why the staining fails.

Regards from Germany
Simone

Offline ImmunoNYC

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adequate counterstaining for DAB
« Reply #5 on: January 21, 2006, 01:21:09 PM »
I think the staining "fails" b/c you are differentiating when you don't need to. It is PROGRESSIVE not REGRESSIVE. Also and perhaps more importantly you are not BLUING adequately. Tap water IS NOT enough.



Quote from: "Simone"
Thank you for your replies and helpful suggestions- new ideas are still welcome...
I use Mayer`s Hematoxylin from MERCK and I already made sure that it is not expired..so there has to be another reason why the staining fails.

Regards from Germany
Simone

Offline apple

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adequate counterstaining for DAB
« Reply #6 on: January 24, 2006, 07:47:45 PM »
I use DAB and Mayer's Hematoxylin. I use tap water to blue nuclei. I get light violet nuclei color and light brown DAB positive color. The photoes looked fine. I can distinguish negative and positive staining very well. I don't think I need to further blue the nuclei.

Offline ImmunoNYC

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adequate counterstaining for DAB
« Reply #7 on: January 24, 2006, 10:31:21 PM »
When quantitating for publications, the nuclei need to be BLUE not VIOLET as blue is further away from brown in the color spectrum than violet ... also I think it yields more attractive photos from an aesthetic perspective. But that's just my opinion of course.

Also, perhaps your tap water is more alkaline than mine and gives you a better bluing. But again when quantitating, reproducibility is fundamental and tap water simply doesn't give me that as it may be different on any given day. :\


Quote from: "apple"
I use DAB and Mayer's Hematoxylin. I use tap water to blue nuclei. I get light violet nuclei color and light brown DAB positive color. The photoes looked fine. I can distinguish negative and positive staining very well. I don't think I need to further blue the nuclei.

Offline apple

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adequate counterstaining for DAB
« Reply #8 on: January 25, 2006, 05:48:33 AM »
Hi, I looked at the website http://www.ihcworld.com/_protocols/histology/bluing.htm. The last solution- Lithium Carbonate Solution. Is it 100ml distilled water or 1000ml distilled water. Could you please have a look?

Thanks




Bluing is also important. Bluing reagent from Richard Allan is a good one. You can also try one of these protocols (http://www.ihcworld.com/_protocols/histology/bluing.htm) and they are working as well.

Offline excalibur

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adequate counterstaining for DAB
« Reply #9 on: January 25, 2006, 10:11:13 AM »
I just use 0.5% ammonium hydroxide to blue.
Paula K. Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
8901 S Santa Fe, Suite G
Oklahoma City, OK 73139
405-759-3959
www.excaliburpathology.com

Offline jkb

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adequate counterstaining for DAB
« Reply #10 on: January 25, 2006, 11:10:51 AM »
Hi,
Are there any chromogens that can be affected by the 0.5% ammonium hydroxide to the blue the counterstain?
Thanks.

Offline excalibur

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adequate counterstaining for DAB
« Reply #11 on: January 25, 2006, 11:15:19 AM »
I have never had a problem with any. It is just a weak alkaline solution.
Paula K. Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
8901 S Santa Fe, Suite G
Oklahoma City, OK 73139
405-759-3959
www.excaliburpathology.com

Offline Simone

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gallery
« Reply #12 on: January 25, 2006, 02:18:03 PM »
To MaximinaNYC:
I would like to see your gallery..where can I find it?
It would be nice if you could send me a link.

Thank you very much,
Simone

Offline ImmunoNYC

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adequate counterstaining for DAB
« Reply #13 on: January 25, 2006, 04:19:27 PM »
If doing IHC on delicate mouse frozen sections the ammonium hydroxide can potentially cause tissue damage. That is why I normally now use commercial bluing solutions. I used to use the ammonium hydroxide until I started doing routine work with frozens. Even so I always keep it on hand for emergencies or when working with paraffin.

Quote from: "jkb"
Hi,
Are there any chromogens that can be affected by the 0.5% ammonium hydroxide to the blue the counterstain?
Thanks.

Offline njcragg

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Counterstaining and DAB
« Reply #14 on: March 07, 2006, 04:39:24 AM »
Just thought I would add our methods to the discussion as it is something I've realised we do differently than the majority.  As image analysis requires a significant distinction between brown and the counterstain colour, we've always used Thionine as a blue counterstain, for better contrast.  We actually use it for all IHC, not just where image anaylsis is employed, because we feel it is a better when doing manual microscopy.
ic

Counterstaining and DAB
« Reply #14 on: March 07, 2006, 04:39:24 AM »