Hello people
I am using HEMATOXYLIN QS (H-3404) from Vector Laboratories for nuclear counterstaining of mice tissues. Unfortunately, in my case this ematoxylin is so strong that removes or covers the brown staining of the DAB. The protocol is the following:
- primary Ab, secondary Ab, Avidin-biotin, incubation with DAB (10 minutes). I checked the DAB staining by microscope, it's good, clear, specific and intense enough.
- block of DAB reaction in water
- 3 drops of hematoxylin directly on the tissues, 10 seconds of incubation,and 5 minutes of running tap water (which has pH=7.5)
- clearing and mounting with xylene
What should I do to have a light staining of hematoxyilin in order to see the brown staining of DAB?
Dear Andrea, The hematoxylin you are using is a modified Mayer's hematoxylin and works very well as a progressive stain for immunohistochemistry techniques. You may control the darkness of the staining in two ways: shortening the time or diluting the solution. I would suggest that you prepare a few consecutive sections (e.g. 6) immunostained for your target antigen (possibly from a paraffin block which is from your current experiments, not particularly useful but representative for your DAB colour intensity) and at the final stage leave them in distilled water. Dilute your H-3404 hematoxylin with distilled water to have 50% and 25% solutions. Stain 2 sections with those 2 concentrations for 5 minutes and process through alcohols and xylene, then coverslip. Do not judge your counterstain at the'wet' stage, as clearing with xylene gives you a much better view if your counterstaining is still too strong. If not satisfied - use the remaining slides to repeat counterstaining with changed timing. Prolong washing after hematoxylin and add a 'bluing' step in an alkaline solution (this contrasts hematoxylin and DAB better). You could also experiment with different different counterstaining e.g. light green. Good luck with your experiments.
To rescue your slides you can remove the coverslip again, go back via xylen, 100% ethanol, 96% ethanol to water. In each reagens move the slides until the surface looks smooth. Then rinse in acidified ethanol for a few seconds until the hematoxylin is gone. DAB will withstand this treatment. After rinsing in water, stain again with diluted hematoxylin and sufficient bluing.
use common differentiators and reduce staining time for haematoxyllin.... also check the water u use to ripen ur H stain... excess salts in water bring higher intensity
Always use deionized/distilled water to wash the slides after hematoxilin staining... usuaslly three washes.
the salts in tap water promote Hematoxilin "maturation"... increasing the darkness of the staining and covering the DAB marking/precipitation.
Hope this helps
Diluting the hematoxylin is the best way to increase the intensity of DAB stain
Dear, I also believe that if you could dilute or reduce the time for incubation with H will give good result. Much better to try this with other or excess tissue preparations and identified the suitable concentrations and incubation time for H counter staining. good luck.
You may removeor weaken the "hematoxylin" stain from an already stained section by a short immersion in slightly acidified 70% ethanol. Selective nuclear staining may be obtained by usin acidified "hematoxylin" solution such as "Mayers acid hemalum".
First I think it will be a good idea using DAB from the Routine lab- a mix of two components. It is dark Brown and stable for more than two days.
Than use a Dilution of 1:2 up to 1:10 of hematoxylin in Aqua bidest. For blueing use a TBS buffer.
And your staining will be much more intensive if you use a modern detection System like polymer or two step polymer without Biotin.
Thanks to all of you guys!! Yesterday I succeeded to have a good hematoxyilin staining!
I just put few drops of hematoxyilin on tissues for 5 seconds and then left the slides in ddH2O (instead of running tap water) for 5 minutes. This was the best protocol that I tested!
Thanks again!
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