The PAS Staining protocol

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5 жыл бұрын

A useful stain for demonstration of polysaccharides (glycogen), neutral mucins and glycoproteins such as those present in basement membranes.

Пікірлер: 29

@RajaRamGurungRaza 3 жыл бұрын

/thank you prof. for your effort to make it understand clearly.

@austinmorrissey5333 2 жыл бұрын

Great video - thank you. Finally I understand why 5 minutes in periodic acid is not working for kidney tissue.

@damienharkin 2 жыл бұрын

Thanks Austin 20 min in Schiff’s reagent will also help. Slightly thinner sections at 2-3 microns will provide sharper details. Good luck!

@Ehem_Ai 2 жыл бұрын

Thank you.

@abdulnoori3315 4 жыл бұрын

that was amazing thank you

@damienharkin 4 жыл бұрын

Thanks Abdul. Let me know if there are any other stains that you would like to see.

@damienharkin 4 жыл бұрын

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

@milogolding1499 11 күн бұрын

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!

@adnanesshakahmed4745 2 жыл бұрын

Thank

@mahjgaming1222 Жыл бұрын

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!

@damienharkin Жыл бұрын

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 KZfaq channel. You pay by the month and can withdraw anytime. Password for the course changes each month. Good luck!

@mahjgaming1222 Жыл бұрын

@@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!

@damienharkin Жыл бұрын

@@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

@damienharkin Жыл бұрын

@@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.

@waelanwar7583 2 жыл бұрын

fuelgen stain reaction plz

@julissamendoza6371 2 жыл бұрын

Excelente pero sería ideal si lo tradujera al español para entender mejor

@hemanthsharma9660 3 жыл бұрын

Hello sir, you did really well, I want to learn something more from you, please help me Sir, understand me

@hemanthsharma9660 3 жыл бұрын

I'm a lab technician

@khinkantkawoo2997 2 жыл бұрын

can I skip --dilute ammonia step ??

@damienharkin 2 жыл бұрын

The ammonia step is necessary to stabilise the bound hematoxylin and turn it blue. Alkaline water can achieve the same result.

@khinkantkawoo2997 2 жыл бұрын

@@damienharkin Thank you.

@julissamendoza6371 2 жыл бұрын

Se puede usar hematoxilina de mayer??

@damienharkin 2 жыл бұрын

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 2 жыл бұрын

@@damienharkin y también puedo usar hematoxilina de Harry ??

@damienharkin 2 жыл бұрын

@@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