Troubleshooting PAS for nonspecific staining

Question 1

Cause: section in PA too long

Answer 1

Resolution/avoidance:

Sulphite rinses after Schiff’s and before water wash- removes unreduced leucofuchsin to prevent non specific reactions

Question 2

Cause: insufficient washing after PA

Answer 2

Resolution/avoidance:

Schiff reagent stored covered in fridge to decrease oxidization

Question 3

Cause: Stronger oxidizer than PA used

Answer 3

Resolution/avoidance:

Prompt fixation for glycogen demonstration

Question 4

Cause: Heavily chlorinated tap water

Answer 4

Resolution/avoidance:

• fresh PA used for each batch of slides
• Schiff not used for more than 2 batches of slides

Question 5

Cause: Fixing in gluteraldehyde - in dialdehyde, one aldehyde group will bind with tissue and the other will be free to react

Answer 5

Resolution/avoidance:

• diastase may life section from slide and is slow acting
• malt diastase may lose some enzyme reactivity above 40degC

Question 6

Cause: Starch paste adhesive on slide will take part in reaction

Answer 6

Resolution/avoidance:
avoid fixing tissues inc chromic acid containing fixatives - variable results but usually overoxidization and weak PAS reaction