Hello. Thanks for the video. We use a blend of both Harris and Gills hematoxylin but have recently been having trouble (literally out of nowhere) where the mucin in our colon tissues or colon biopsies are staining blue and in some cases the nucleus and nuclear detail is too blue/purple. How can we get rid of this so th ey look more like your first example I’m this video …?
Hi Cristina, The outcome that you describe indicates insufficient differentiation in acid alcohol following application of Hx. I would suggest using fresh acid alcohol or increasing your time in acid alcohol. To be sure, you should examine your slide by microscope following each round of differentiation and subsequent blueing. Hope this helps. Let me know how it goes! Bye for now Damien
Thanks for this video, Sir I want the interpretation for my lab work, but am feeling like I need a help of a good histopathologic can you help me with that?
excuse me, I want to ask something. If we want to observe the histopathology of specific inflammatory cells such as macrophages, neutrophils, and lymphocytes in mouse skin tissue, is it enough to just use HE staining? or should use immunohistochemical staining?
Good question. While the basic morphological characteristics of different leukocytes can often be seen quite well in H&E stained sections (e.g., polymorphonuclear leukocytes versus monocytes and lymphocytes), immunohistochemistry enables specific subsets of leukocytes to be demonstrated (e.g., CD4 versus CD8 positive lymphocytes).
i guess im asking the wrong place but does anyone know a method to log back into an instagram account..? I somehow lost my account password. I love any help you can offer me!
Hi Ravi, we use a number of different hematoxylin recipes but mainly use Ehrlich's when performing H&E stains. You can buy from a commercial supplier but we prepare as follows. Haematoxylin: 16 g Absolute Ethanol: 480 mL Aluminium Potassium Sulphate: 48 g De-ionised water: 240 mL Glycerol: 240 mL Glacial Acetic Acid: 24 mL Dissolve the haematoxylin in alcohol using gentle heat. Dissolve the Aluminium Potassium Sulphate in water in the same way and whilst still warm add the glycerol. Allow to cool. Add the haematoxylin solution in small portions and mix well. Finally add the acetic acid. Plug the container with cotton wool or other loose stopper and allow to ripen by exposure to light, this will take 4 6 weeks but may be allowed to continue indefinitely. Filter before use (e.g. Whatman No. 1 filter paper). If necessary, more immediate oxidation can be achieved by adding 0.1g of Sodium Iodate per 100 mL of stain. Allow to stand for 1hr prior to use. Our eosin solution is prepared by combining the following but can also be purchased commercially. Absolute Alcohol: 444ml De-ionised water: 24ml 1% aqueous Eosin Y: 60ml 1% aqueous Phloxine: 1.5ml Glacial Acetic Acid: 2.4ml Good luck with your staining! Damien
Good morning professor my slid didn't take hematoxylin i leave inside it for 15 minute 😢why? This is how i do staining Oven 90 for 1 hours 3 xyline each 15 minute 3 Alcohol each 10 minute Water 10 minute Hematoxylin 15 minute Water Eosin 3 alcohol each 1 minute Pleas please help me😢 Any one see my comment pleas help
Hello. 15 minutes should certainly be long enough to see some staining with Hx. Are you preparing the Hx yourself or buying from a supplier. Assuming that the Hx Solution is fine, it is possible to remove if treat for too long in acid alcohol (ie. if using a regressive staining technique), but you don’t make mention of using this. Another possibility is that slides have not been dewaxed sufficiently to allow entry of stains, but this seems unlikely given your time in xylene. Does the eosin stain the tissue at all?
Another possibility is that you are heating your slides at 90 degrees rather than 60-70 degrees C. This should be ok, but just something to consider in case causing some damage to sections. I also assume that the tissue has been initially fixed and processed correctly.
Hello. Thanks for the video. We use a blend of both Harris and Gills hematoxylin but have recently been having trouble (literally out of nowhere) where the mucin in our colon tissues or colon biopsies are staining blue and in some cases the nucleus and nuclear detail is too blue/purple. How can we get rid of this so th ey look more like your first example I’m this video …?
Hi Cristina,
The outcome that you describe indicates insufficient differentiation in acid alcohol following application of Hx.
I would suggest using fresh acid alcohol or increasing your time in acid alcohol.
To be sure, you should examine your slide by microscope following each round of differentiation and subsequent blueing.
Hope this helps. Let me know how it goes!
Bye for now
Damien
Great work sir!
Glad you liked it!
Thanks for this video, Sir I want the interpretation for my lab work, but am feeling like I need a help of a good histopathologic
can you help me with that?
Hi Sana,
I’m not a pathologist, but can provide advice on histological staining techniques to client level members of my RUclips channel.
excuse me, I want to ask something. If we want to observe the histopathology of specific inflammatory cells such as macrophages, neutrophils, and lymphocytes in mouse skin tissue, is it enough to just use HE staining? or should use immunohistochemical staining?
Good question. While the basic morphological characteristics of different leukocytes can often be seen quite well in H&E stained sections (e.g., polymorphonuclear leukocytes versus monocytes and lymphocytes), immunohistochemistry enables specific subsets of leukocytes to be demonstrated (e.g., CD4 versus CD8 positive lymphocytes).
thank you so much. Just in time for my exam! :)
i guess im asking the wrong place but does anyone know a method to log back into an instagram account..?
I somehow lost my account password. I love any help you can offer me!
@Decker Jadiel instablaster ;)
Hi, can we reverse overdifferentiated Hx before putting in eosin?
Yes. Just stain more with Hx.
Thank you sir.
Dear sir..can you please tell me the Preparation of Hematoxylin and Eosin
Hi Ravi, we use a number of different hematoxylin recipes but mainly use Ehrlich's when performing H&E stains.
You can buy from a commercial supplier but we prepare as follows.
Haematoxylin: 16 g
Absolute Ethanol: 480 mL
Aluminium Potassium Sulphate: 48 g
De-ionised water: 240 mL
Glycerol: 240 mL
Glacial Acetic Acid: 24 mL
Dissolve the haematoxylin in alcohol using gentle heat. Dissolve the Aluminium Potassium Sulphate in water in the same way and whilst still warm add the glycerol. Allow to cool. Add the haematoxylin solution in small portions and mix well. Finally add the acetic acid. Plug the container with cotton wool or other loose stopper and allow to ripen by exposure to light, this will take 4 6 weeks but may be allowed to continue indefinitely. Filter before use (e.g. Whatman No. 1 filter paper).
If necessary, more immediate oxidation can be achieved by adding 0.1g of Sodium Iodate per 100 mL of stain. Allow to stand for 1hr prior to use.
Our eosin solution is prepared by combining the following but can also be purchased commercially.
Absolute Alcohol: 444ml
De-ionised water: 24ml
1% aqueous Eosin Y: 60ml
1% aqueous Phloxine: 1.5ml
Glacial Acetic Acid: 2.4ml
Good luck with your staining!
Damien
@@damienharkin Thank you so much sir.....
where are you in UK or US
Brisbane, Australia.
Good morning professor my slid didn't take hematoxylin i leave inside it for 15 minute 😢why?
This is how i do staining
Oven 90 for 1 hours
3 xyline each 15 minute
3 Alcohol each 10 minute
Water 10 minute
Hematoxylin 15 minute
Water
Eosin
3 alcohol each 1 minute
Pleas please help me😢
Any one see my comment pleas help
Hello. 15 minutes should certainly be long enough to see some staining with Hx. Are you preparing the Hx yourself or buying from a supplier. Assuming that the Hx Solution is fine, it is possible to remove if treat for too long in acid alcohol (ie. if using a regressive staining technique), but you don’t make mention of using this. Another possibility is that slides have not been dewaxed sufficiently to allow entry of stains, but this seems unlikely given your time in xylene. Does the eosin stain the tissue at all?
Another possibility is that you are heating your slides at 90 degrees rather than 60-70 degrees C. This should be ok, but just something to consider in case causing some damage to sections. I also assume that the tissue has been initially fixed and processed correctly.
👍