Microscope tutorial - troubleshooting PAS stains

[@6202kamel]

A very good description, thank you so much for doing such a great job

[@damienharkin]

Many thanks. Hope to upload some more videos soon.

[@goharrehman7710]

we need to have some about fixative and about antigen or antibodies IHC staining please

[@damienharkin]

Ok. Can do one comparing the effects of different fixatives and another demonstrating typical immuno-histochemistry outcomes.

[@Apoptotickitler]

Thanks for the video! I counter stain my pas with my hematoxylin, however, it is very dark and i end up with my slides fully in purple and i can't really see the pink from PAS stainig. I am doing hematoxylin for 90 seconds as stated in the protocol kit, what could be the problem?

[@damienharkin]

Hi Malay Try staining for smaller increments of 15-20 seconds, then rinse, blue with ammonia, then check level of staining. Repeat if necessary until reach desired level of staining. I typically use 20-30 seconds for Mayers’s Hematoxylin with PAS stain. Let me know how you go.

[@Apoptotickitler]

@@damienharkin I will let you know when i try next week. I don't think i have ammonia in the lab. I have read that sodium bicarbonate can work as an alternative because the purpose is to slightly increase the ph of the water ? Can i just use tap water and increase the PH with drops of NaOH?

[@damienharkin]

Yes, but don't add too much NaOH. Solution just needs to be slightly alkaline between 7.5-9.0.

[@RavenclawSeer]

Thank you. Have you done a video on Mucicarmine or amyloid, or an Alcian blue stain? Basically the carbohydrates stains?

[@damienharkin]

We cover all these stains in our classes at QUT. Just haven't got around to producing a video yet. Carbohydrate chemistry gets pretty complex but the staining protocols are fairly straight forward. Hope to post something within the next few months.

[@RavenclawSeer]

@@damienharkin Thank you so much for replying. Yes, carbohydrates chemistry is very complicated to me. But I admit, as a student, the stains seem straight forward. I appreciate your work!

[@NicksLocker]

Thank you very much!

[@damienharkin]

My pleasure.

[@19turan]

Would be able to demonstrate Oil Red O stain? Thank you in advance

[@damienharkin]

Great suggestion!

[@sarangas8688]

What are the precaution takes when doing pas stain ?

[@damienharkin]

Make sure that your Schiff's reagent is stored refrigerated, rinse in distilled water prior to applying Schiff's (to reduce background) and adjust your staining times to suit the target being stained (e.g. Treat for 20 minutes if staining basement membranes. Oh, and finally, always rinse well in water after treating with Schiff's reagent. Hope that helps.

[@sarangas8688]

@@damienharkin thank you so much 😊😊

[@nearby119]

What if periodic acid is left for too long? What will happen and what can we to counteract it?

[@damienharkin]

Great question! Theoretically periodic acid is not a strong enough oxidising agent to convert the Di-aldehyde groups into carboxylic acid groups, so there’s no harm if leave on for longer.

[@gowrimahi8920]

Thank you sir ji

[@user-xk9pq5px9h]

Hi! Thank you for this video. I am busy trying to optimize PAS staining of mouse brain, lung and spleen sections to visual cryptococcus neoformans infection. However, I find that my sections stain very dark purple so I can't see the fungi. I was thinking of reducing the time of the schiffs stain (currently 15min) and reducing the time of the wash step after schiff staining (I've noticed this step intensifies the dark purple). Do you have any other suggestions? Thanks!

[@damienharkin]

I would suggest reducing the time in Schiff's reagent to 10 minutes. The rinsing in water is essential for generating colour (releases the unstable sulfonic acid group), but 5 minutes should be sufficient. Also, make sure that your sections are not too thick. Would suggest use 3 µm sections. Hope this helps. I provide examples of staining for cryptococcus using the PAS method in my online course "Essentials of Histological Technique". You can access the course by joining as a Trainee Member of my KZbin channel.

[@pratishthamadaan1038]

More videos pls

[@damienharkin]

Hi Pratishtha, Is there anything in particular that you would like to see demonstrated?

[@gowrimahi8920]

Can I get principle application and procedure PAS