Hi PAS experts, I am trying to use the PAS stain for glycogen assessment in the liver of fish. However, when I perform the stain, it seems to be a pretty intense and dark purple as compared to the lighter purples/magenta I see other stains having. I am using the Sigma-Aldrich PAS staining kit (1016460001) and the protocol I am using is as follows:

1. 30s dH2O

2. 5* mins Periodic Acid

3. 3 mins running tap water

4. 30s dH2O

5. 15* mins Schiff's

6. 5 mins running tap water

7. 30s dH2O

8. 30s* Gill's no.1

9. 3 mins running tap water

10. dehydrate and mount

I have tried changing the time AND dilution of haematoxylin, as I noticed it was making the purple more intense. I even tried it without counterstain, and messed with the periodic acid times (5 vs 10 mins) and Schiff's (5, 10, 15 mins). However, regardless of what I do, the stain comes out very purple. I have attached some pictures to illustrate. Please let me know if there is anything I can do to lighten the purple (or if what I'm seeing is actually correct...), as currently, even if I counterstain with a dilute Gill's, it can't be seen.

Thank you so much!

The images attached are:

1. with normal concentration of gills

2. with a 25% gills in dH2O

3. no counterstain