Professor Harkin, in the experiment, did you use pure hematoxylin or a dilution of it? If it was a dilution, what was the dilution ratio that you used? Thank you for your wonderful video!
Thanks for the wonderful video. I was wondering if you could give me some guidance. I'm having issues performing the pas stains on fish liver to assess glycogen, the stain turns out a very dark purple/blue hue instead of the nice pink hues I see in most literature. My protocol is as follows, and for parts I've tried to adjust I've starred: 1. 30s dh2o 2. 0.5% PAS for 5 mins 3. 3 min running water (tap) 4. 30s dh2o 5*. Schiff for (15, 10, 5 mins) 6. 5 min running water (tap) 7. 30s dh2o 8*. Gills no.1 for (5, 3, 1, 30s, 1 dip) I thought the purple and blue hue might have been from the Gill's. I also tried 1 minute of ehrlichs haematoxylin which did result in less "blue" but I couldn't see the nuclei too well. 9. Running water for 3 mins (tap) 10. Dehydration (95% then 100%) then clear and mount Do you think it's the haematoxylin that's causing the dark purple/blue colour? Or is it perhaps not a long enough pas or Schiff step? I thought it may have been in Schiff for too long that's why I tried decrementing the time in Schiff's, but no luck. Thank you!
Are you able to check the slide during staining? E.g. after each staining step? It does sound like the Hx is too strong, but I would check on level of glycogen staining first. BTW-you can access my 18 part online course now by joining as either a “trainee” or “client” level member of my TH-cam channel. You pay by the month and can withdraw anytime. Password for the course changes each month. Good luck!
@@damienharkin Thanks for the reply. I ended up trying no counterstain and it seems like while the Hx did affect it, the PAS alone was still quite purple. I tried increasing Periodic Acid Solution time but it made the PAS stain even more intense (makes sense, I suppose). Really at a lost for how to lighten the PAS. :( Also thanks, did not know!
@@mahjgaming1222 glycogen can be quite intense when stained but your description of colour doesn’t sound right. Please send me some pictures if able via email to d.harkin@qut.edu.au
@@mahjgaming1222 Your problem might also be related to section thickness. If you have a lot a glycogen, the PAS staining outcome will be very intense if your sections are too thick. I would recommend cutting sections around 3 µm.
Yes, Mayer’s hematoxylin is normally used as the counter stain following application of PAS. 15-20 seconds is usually enough staining time, followed by “bluing” in dilute ammonia.
@@julissamendoza6371 I haven’t tried Harris Hx after PAS, but I think it should still work. I recommend reducing the staining time however from 10 to 3-5 minutes since the periodic acid (oxidation) increases binding of Hx to the tissue. Let me know how it goes. Can send me a picture at d.harkin@qut.edu.au
Hi Ahsen, I'm planning a special series on silver stains so GMS will be covered eventually. Mucicarmine will also be covered as part of additional stains for carbohydrate. Bye for now. D
/thank you prof. for your effort to make it understand clearly.
Great video - thank you. Finally I understand why 5 minutes in periodic acid is not working for kidney tissue.
Thanks Austin
20 min in Schiff’s reagent will also help. Slightly thinner sections at 2-3 microns will provide sharper details.
Good luck!
Professor Harkin, in the experiment, did you use pure hematoxylin or a dilution of it? If it was a dilution, what was the dilution ratio that you used? Thank you for your wonderful video!
We use our Mayer’s Hx undiluted for around 10-15 seconds following application of PAS stain, then “blue” with dilute ammonia.
Thanks for the wonderful video. I was wondering if you could give me some guidance. I'm having issues performing the pas stains on fish liver to assess glycogen, the stain turns out a very dark purple/blue hue instead of the nice pink hues I see in most literature.
My protocol is as follows, and for parts I've tried to adjust I've starred:
1. 30s dh2o
2. 0.5% PAS for 5 mins
3. 3 min running water (tap)
4. 30s dh2o
5*. Schiff for (15, 10, 5 mins)
6. 5 min running water (tap)
7. 30s dh2o
8*. Gills no.1 for (5, 3, 1, 30s, 1 dip) I thought the purple and blue hue might have been from the Gill's. I also tried 1 minute of ehrlichs haematoxylin which did result in less "blue" but I couldn't see the nuclei too well.
9. Running water for 3 mins (tap)
10. Dehydration (95% then 100%) then clear and mount
Do you think it's the haematoxylin that's causing the dark purple/blue colour? Or is it perhaps not a long enough pas or Schiff step? I thought it may have been in Schiff for too long that's why I tried decrementing the time in Schiff's, but no luck. Thank you!
Are you able to check the slide during staining? E.g. after each staining step?
It does sound like the Hx is too strong, but I would check on level of glycogen staining first.
BTW-you can access my 18 part online course now by joining as either a “trainee” or “client” level member of my TH-cam channel. You pay by the month and can withdraw anytime. Password for the course changes each month.
Good luck!
@@damienharkin Thanks for the reply. I ended up trying no counterstain and it seems like while the Hx did affect it, the PAS alone was still quite purple. I tried increasing Periodic Acid Solution time but it made the PAS stain even more intense (makes sense, I suppose). Really at a lost for how to lighten the PAS. :(
Also thanks, did not know!
@@mahjgaming1222 glycogen can be quite intense when stained but your description of colour doesn’t sound right. Please send me some pictures if able via email to d.harkin@qut.edu.au
@@mahjgaming1222 Your problem might also be related to section thickness. If you have a lot a glycogen, the PAS staining outcome will be very intense if your sections are too thick. I would recommend cutting sections around 3 µm.
Thank you.
Se puede usar hematoxilina de mayer??
Yes, Mayer’s hematoxylin is normally used as the counter stain following application of PAS.
15-20 seconds is usually enough staining time, followed by “bluing” in dilute ammonia.
@@damienharkin y también puedo usar hematoxilina de Harry ??
@@julissamendoza6371 I haven’t tried Harris Hx after PAS, but I think it should still work.
I recommend reducing the staining time however from 10 to 3-5 minutes since the periodic acid (oxidation) increases binding of Hx to the tissue.
Let me know how it goes. Can send me a picture at d.harkin@qut.edu.au
can I skip --dilute ammonia step ??
The ammonia step is necessary to stabilise the bound hematoxylin and turn it blue. Alkaline water can achieve the same result.
@@damienharkin Thank you.
that was amazing
thank you
Thanks Abdul. Let me know if there are any other stains that you would like to see.
Hi Ahsen, I'm planning a special series on silver stains so GMS will be covered eventually. Mucicarmine will also be covered as part of additional stains for carbohydrate. Bye for now. D
Excelente pero sería ideal si lo tradujera al español para entender mejor
Hello sir, you did really well,
I want to learn something more from you, please help me Sir, understand me
I'm a lab technician
fuelgen stain reaction plz
Can i get theory notes
The theory notes can be accessed by joining my channel as a “trainee histologist” level member.
Thank
Sir or bhi staning ki video dailye
Trying to speak clear sir