There are different types of Hematoxyline, and depending on the type of Haematoxyline (Mayer's Haematoxylin, Harris's Haematoxylin, Haidenhain's Haematoxylin, Mallory's Phosphotugustic Acid Haematoxylin and Iron Haematoxylin).

As I have taught the courses to undergraduate and graduate students on histological and histochemical techniques (more than 30 years) I can explain why such precedure was used.

I assumed that the procedure being mentioned here is for the Mayer's Hematoxylin stain. As some people already wrote tap water wash is sufficient and it is true, especially if the tap water is a hard water (it is alkaline and colour of Hematoxyline turns bluish in tap water and it is more natural and the colour doesn't fade after a long term storing of the slides). HCL causes the colour to become lighter and this process is being used in Iron Haematoxylin stain in which we over-stain the tissue and remove the excess colour with HCL and this procedure is called differentiation.

There are many labs that use Ammonium Hydroxyde for making the colour blue (from purplish) quickly but tap water is more natural and gentle on the tissue.

Hi Abhilash

I have never used this solution for washing hematoxylin stain. I always wash Hematoxylin with tap water (also in order to activate the stain). The important things are: the type of the tissue, the hematoxylin dilution, the exposing time to the hematoxylin. What kind of hematoxylin you are used to use? Is it pure or diluted ? And what kind of sample ?

I completely agree, we use tap water to...

Hi Abhilash,

After washing Hematoxylin with tap water we dipped twice in HCl 1% in destillated water and we washed again in tap water (we don't use ammonium hydroxide). If you don't have HCl the only solution I have is to incubate less time with the Hematoxylin or to use it diluted...

Hi Abhilash

please, i advise you to read about regressive and progressive stains. Also, try to adjust the staining time by dipping many slides in the stain for different times (ex. 2, 3, 4, and 5 minutes) and examining the stain under the light microscope. I prefere to wash the slides in DW, then dip the staining slides in acid alcohol 2 to 3 times. Finally, wash the slides in running tap water for 5-7 minutes. Good luck

Yes I too use tap water.

Thanks all. I will take your suggestions.

We wash the slide in DW and then observe the same under microscope. In case of overstaining washing in acid water is done. However, it is always preferable to stain the slides in haematoxylin for not more than 3 minutes and then dipping it in DW for 5 to 8 minutes. Try it,hope u will get satisfactory result.

There are different types of Hematoxyline, and depending on the type of Haematoxyline (Mayer's Haematoxylin, Harris's Haematoxylin, Haidenhain's Haematoxylin, Mallory's Phosphotugustic Acid Haematoxylin and Iron Haematoxylin).

As I have taught the courses to undergraduate and graduate students on histological and histochemical techniques (more than 30 years) I can explain why such precedure was used.

I assumed that the procedure being mentioned here is for the Mayer's Hematoxylin stain. As some people already wrote tap water wash is sufficient and it is true, especially if the tap water is a hard water (it is alkaline and colour of Hematoxyline turns bluish in tap water and it is more natural and the colour doesn't fade after a long term storing of the slides). HCL causes the colour to become lighter and this process is being used in Iron Haematoxylin stain in which we over-stain the tissue and remove the excess colour with HCL and this procedure is called differentiation.

There are many labs that use Ammonium Hydroxyde for making the colour blue (from purplish) quickly but tap water is more natural and gentle on the tissue.

I usually use tap water with a rinse in distilled water. If your sections are poorly attached to the slide be careful not to lose them in tap water.

Good luck

Based on what you are saying about "extra color" to the tissue sections it sounds as if yo are doing Harris Hematoxylin staining where you over-stain the tissue and use a differentiation step after the bluing step. Tissue is 'blued' by either running in tap H2O or with a commercially prepared solution such as Scott's Bluing Solution. The differentiation step is achieved by acid alcohol (0.4% HCL in EtOH). If you are using a progressive method such as Mayer's Hematoxylin, reduce your staining time in the Hematoxylin to reduce the extra color. I use Mayer's Hematox. for 5min with excellent results, NO differentiation!

Hi

What haematoxylin are you using. For Mayers and Harris's Haematoxylin these are used as regressive stains i.e you overstain the sections then remove some of the haematoxylin using 1% hydrochloric acid in 70% ethanol to reveal the nuclear components (this is differentiation). You should always control this step by examining the sections microscopically so as not to overdifferentiate i.e remove too much of the nuclear stain but if this occurs you can go back into haematoxylin. Using an alkaline solution to 'blue' the nuclei should be done separately. If you are in a hard water area tap water will do this for you or you could use Scotts tap water. Hope this helps.

The most recent answer by Dr. Jessica Mainez doesn't say for which Hematoxylin.

For Mayer's Hematoxylin, the nuclei are stained in reddish colour and in order to change the colour to blue, you need an alkaline solution such as tap water (hard water is better) or Ammonium Hydroxide. For Harris's Hematoxylin, Ammonium Hydroxide is not needed. As you must know that Eosin stains intensely in low pH whereas Hematoxylin stains better in alkaline pH.

If you are a histologist or histopathologist you want to find out the tissue component and it is important to assess acidophilia and basophilia of the cells and tissues. We must consider the property of the dyes and their binding (staining) mechanism and standardize the precedure.

If you use Weigerts hematoxylin, the nuclei turn more black in color and do not need to go through the HCL/ ammonium steps. Instead you would just wash under running water for 10 minutes. If you are using Harris hematoxylin, then instaed of using ammonium hydroxide you can use saturated lithium carbonate solution. 1.0gm lithium to 100.0ml of DH2O.

Rinse in ethanol 30% to 50%

I think this information may fulfill your requirement.

Blueing is done with alkaline solutions such as hard tap water (pH 8 to 8.4), Scott’s tap water substitute, 0.1% ammonia water, 1% sodium acetate, 0.5% lithium carbonate etc.

You are right Mr. Abhilash D. Pandya, I also faced this type of problems. I will suggest below steps,

1) If you are using Mayer’s Haematoxylin Need to stain 2 – 3 mints (or)

2) If you are using Harris’s Haematoxylin need to stain only 10 sec,

then wash (both 1 & 2) in running tap water from reverse side. Finally rinse in PBS for 5 minutes (No need to do differentiation process). Try it,hope u will get satisfactory result.

We usually use tap water and have good result.Some use the below procedure and claim to have good results(you can try):

Staining procedure

1. Dewax and bring to water

2. Stain in Mayer's hematoxylin* 8 min.

3. Rinse briefly in tap water

4. Differentiate in HCl-alcohol 0.5% 3x brief

5. Rinse in tap water and blue 10 min.

6. Erythrosin(A 1%) 2 min.

7. Rinse briefly in tap water

8. Dehydrate through alcohols, clear in xylol

9. Mount sections as usual

i use 1% HCl solution in 70% ETOH to avoid over hematoxylin staining for 30-60 seconds.

OK

I think all answers are quite similar and very useful, thanks to all who contributed and Dear Abhilash now you can try any of the suggested answers depending on what type of haematoxylene you are using. Please let us know whatever results you obtain

Here's your protocol:

H&E

1. Dewax and bring to water

2. Stain in Mayer's hematoxylin* 10 min.

3. Wash in tap water 5 min

4. Differentiate in HCl-alcohol 1% in 70% alcohol 25 seconds

5. Wash in tap water 30 seconds.

6. Rinse in lithium carbonate (saturated solution) for 30 Seconds (bluing solution)

7. Wash in tap water 3 minutes

8. Alcohol 70% 2 min

9. Alcool 80% 2 min

10. Eosin (alcoholic preparation) 1 min

11.Alcohol 96% 2 min

12.Alcohol 96% 2 min

13. Alcohol 100% 2 min

14. Alcohol 100% 2 min

15. Xylene and mount with DPX

Hope this helps!

Best Regards

As Dr.Shigeto Yamashiro mentioned,there are different types of Haematoxylin formula depending on the requirement whether you are doing regressive or progressive staining. For tissue studies(Histopathology), progressive staining method is often used with Mayers hematoxylene. Here the tissue is stained for a desired time(2 to 3 minutes) Many people use this method without out rinsing the tissue in in HCl(0.05%-1%) and bluing in running water. Differentiation is often done in regressive method of staining using dilute HCl(0.05-1%).This method is often followed in cytology samples where nucleus is over stained and the differentiation gives more vesicularity to the nucleus,clearing the parachromatin area.Also it helps to clear hematoxylin from the cytoplasm.Bluing is done in an alkaline solution(ammonical water,lithium carbonate solution) or running tap water for both of the technique.

How about using lithium carbonate solution ?

Dear Dr. Seshadri Jagannathan,

Lithium carbonate solution can be used as a bluing agent.But it is known to cause floating of cells and tissue materials from the slide.

I usually use 1% HCl solution in 70% ETOH depending of hematoxylin solution efficiency if it was fresh I rinse in 1% HCl solution in 70% ETOH for 3 sec but it was old and turned to brown I rinse slides one sec in 1% HCl solution in 70% ETOH or even delete this stsge.

My protocol to stain with H&E

1. Dewax and bring to water

2. Stain in hematoxylin 5-8 min.

3. Wash in tap water 2-3 min

4. Differentiate in HCl-alcohol 1% in 70% alcohol 25 seconds aprox.

5. Wash in tap water 30-50 seconds.

6. Rinse in lithium carbonate (saturated solution) for 50 Seconds (bluing solution)

7. Wash in tap water 2 minutes

8. Wash distilled water

9. Alcool 96% 1 min

10. Eosin (alcoholic preparation) 35-50 seconds

11.Alcohol 96% 2 min

12.Alcohol 96% 2 min

13. Alcohol 100% 2 min

14. Alcohol 100% 2 min

15. Xylene-I 5-8 min.

15. Xylene-II and mount with Cytoseal 60

Best Regards

washing with 0.1% HCl is enough for post washing step of HX stain

Not anybody here in this posting interesting thread, but an informative interposed question:

Doesn't (solution) have an other meaning than " solution"?

(Google: < bluing or blueing solution? > )?

Or is the word used really frequently in ? Perhaps a native speaker can reply to my honestly posted question... Thanking you in advance,

Wolfgang

They are interchangable in the English language. Sometimes you will even see it spelled 'bleuing', but the most recognized spelling is 'bluing'.

First off the Ammonium hydroxide is not to differentiate the Hematoxylin, i.e. remove color, your acid alcohol does that. Ammonium hydroxide is used to blue the nuclei after removal of the excess hemalum by the weak acid. Try a progressive method that does not need a differentiation step. Mayer's Hematoxylin (polyscientific R&D Co.) Let me know if you want my protocol.

Beth