3. •
Methods for glycogen staining
•
PAS technique
•
Principle
•
What is Schiff’s reagent
•
Procedure
•
comment
•
Alternative techniques
•
Diagnostic applications
•
How to make a negative control slide
5. Periodic acid Schiff's tech. (PAS)
•
It is the most widely method used for the
demonstration of carbohydrate(glycogen or
mucin).
•
PAS tech. may aid in the differential diagnosis of
tumor through the detection of mucin or
glycogen.
•
PAS tech. based upon broken of 1.2 glycol group
by oxidation and convert it to aldehyde
group(within CHO) which react with Schiff’s
reagent and produce a bright red magenta color.
6. Principle of PAS
•
Initial step in PAS is the oxidation of 1.2glycol
group(in the CHO), which result is the
formation of free aldehyde group, that react
with Schiff's reagent and produced magenta
color.
7. principle of PAS
•
1-glycol group + periodic acid(oxidation)=
•
free aldehyde group
•
2-Free aldehyde group + Schiff's reagent=
magenta color
8. Schiff’s reagent??
•
It’s a colorless leucobases dye, its
chromophoric group was destroyed by
sulpheration
•
It’s clear or pale yellow in color
9. Solutions of Schiff's reagent
•
1- basic fuchsine: true dye contain
chromophore group
•
2- sodium or potassium meta bisulphate :
sulpheration ( destruction for the
chromophore group in acidify media )
•
3- hydrochloric acid : give acidify media
•
4- charcoal : color absorption
•
5- D.W : solvent
10. Principle of Schiff's reagent
chromophore group “quinoid” structure in the
basic fuchsine destructed by sulpheration(Na
or K met bisulphate) and convert the dye to
colorless dye, reconstitution of the
chromophore group done when the free
aldehyde group react with the Schiff's reagent
gives magenta color.
11. Preparation of Schiff's reagent
•
Dissolve 1g of basic fuchsine and 1.9g of
sodium meta bisulfate in 100ml of 0.15 N
hydrochloric acid.
•
Shake the solution at intervals for 2hr.
•
Add 500mg of activated charcoal and shake
for 1-2min.
•
Filter the solution through Whatman filter
into a bottle, the solution should be colorless
or yellow in color, then store it at 4c.
12. Procedure of PAS
•
1-deparaffinize in xylene 6-8 min
•
2-rehydrate in Abs. , 90% alcohol, 70% alcohol, D.W
2 min in each one
•
3-oxidize with periodic acid for 5 min
•
4-rinse in D.W
•
5-treat section with Schiff’s for 15-30 min
•
6-rinse in running tap water for 5-10 min
•
7-stain nuclei with Mayer's for 5-7 min
•
8-bluing in running tap water for 10min
•
9-dehydrate by alcohol, clear by xylene, mount by D.P.X
16. Diagnostic applications
•
Glycogen may demonstrated in a number of
lesions and tumors such as:
•
Carcinoma of bladder, kidney
•
Adeno carcinoma of the ovary and lung
•
Juvenile rhabdomyosarcoma
•
Seminoma
•
Ewing’s sarcoma
•
Glycogen storage disease
17. Negative control for glycogen
•
Enzymatic methods :
•
1-alpha amylase: hog pancreas
•
2- beta amylase: sweet potato
•
3- diastase: malt, contain both alpha and beta
amylase
4-saliva enzyme : (37c for 1hr in water bath)
•
principle: these enzymes act on glycogen, convert
it into smaller sugar units( maltos&glucose)
