Staining in Histopathology, special stains used in the laboratory

1. Histopathology II Practical
Course code: MLS 311

2. SPECIAL STAINS FOR
CARBOHYDRATES

3. Objectives
By the end of the practical session, students must be able to
• Name and describe various special stains for carbohydrates
• State the indications for staining procedure
• State the principles of staining technique
• Outcomes of staining technique

4. Recap:
• What are carbohydrates?
• Name the types of carbohydrates
• What is the importance of carbohydrates to the tissues?
• Name some of the complex carbohydrates.

5. Stains to be considered for staining
carbohydrate complexes
• Periodic Acid Schiff (PAS) for Neutral and mixed mucins
• Alcian blue for acid mucins
• Diastase Digestion Method
• Alcian Blue- PAS
• Congo Red (Bennhold`s) method for Amyloid
• Crystal violet (Lieb`s) Method for Amyloid

6. Periodic Acid Schiff (PAS)
• PAS demonstrates neutral polysaccharides present in the
basement membrane and the secretion of various glands in
our body
• Widely used in histopathology to demonstrate glycogen,
fungi and mucin.

7. Indications
• To demonstrate polysaccharides:
• Glycogen= glycogen storage disorders
• Cellulose
• Starch
• Glycoproteins
• Glycolipids
• Pigments
• Plasma cells

8. Principle
• Hydroxyl group of Carbohydrate molecule is oxidized to the
aldehyde CHO group by periodic acid
• CHO reacts with Schiff’s reagent to form a magenta coloured
compound

9. Preparation of solutions
• Solution 1:
• Periodic acid= 1g
• Distilled water= 100ml
• Solution 2 (Schiff’s reagent)
• Basic fuchsin (CI #. 42510) = 1g
• Distilled water = 200ml
• 1M HCl = 20ml
• Potassium bisulphite = 2g
• Activated charcoal = 0.5g

10. Preparation of solution 2
• Measure distilled water into a 500ml flask
• Boil distilled water
• Dissolve basic fuchsin.
• Cool to 50℃
• Filter and add HCl
• Cool to 25℃ and add sodium bisulphite
• Store in the dark for 24-48hrs during which time the solution becomes
straw coloured
• Shake up with the charcoal and filter immediately
• Transfer filtrate to a brown bottle and label.
• Store in a refrigerator

11. Solution 3
• Sodium metabisulphite ,10% = 6ml
• 1M HCl = 5ml
• Distilled water = 100ml
Solution 4
• Harris alum-haematoxylin

12. Staining procedure
1. De-wax tissue and hydrate tissue
2. Place in solution 1 for 5 - 10mins
3. Rinse in tap water
4. Rinse in distilled water
5. Place in Schiff’s reagent for 5 mins
6. Place in 3 baths of solution 3 for 2 mins in each bath
7. Rinse in tap water
8. Counter stain with solution 4 for 30 secs
9. Blue in tap water
10. Dehydrate ,clear and mount

13. Results
• Cell nuclei = blue
• Mucin and glycogen = Purple
• Basement membrane of kidney and skin = Reddish purple

14. Quality control measures
• Add drops of Schiff’s reagent to formalin
• Active Schiff’s reagent should quickly change the colour of formalin to
pink

15. Quality control
• Always have positive and negative control samples for daily quality
check for staining
• Remember to document results of quality control

16. Pictures

17. ALCIAN BLUE

18. Introduction
• A large conjugate dye molecule initially used for dyeing textiles fibres
• Alcian blue stains acid mucins (at pH 2.5)
• Stains mucins of salivary glands, prostate, and large intestine
• It also stains proteoglycans in cartilaginous material

19. Indications
• Intestinal metaplastic cells in Barrett’s oesophagus and stomach
biopsy
• Mucinous adenocarcinoma of the ovary
• Pleural mesothelial cells
• Myxomas
• Mucinous material in myxomas: Discoid Lupus Erythematosus lesion

20. Principle
• Alcian blue is a group water soluble polyvalent basic dyes
• Dye made of copper-containing phthalocyanine ring with the copper in the middle
• Phthalocyanine is also attached to four isothiouronium groups that are positively
charged.
• Positively charged alcian blue dye complex has an attraction for anionic sites on mucin.
• Carboxylated and sulfatic proteoglycans are stained at pH of 2.5
• Copper impacts blue colour of the dye-mucin complex

21. Stain preparation
• Alcian blue solution
• Alcian blue, 8GX 1% aqueous solution : 1g
• Glacial Acetic acid (3%) solution : 100ml
• Neutral red solution
• Neutral fast red : 1g
• Aluminium sulphate : 5g
• Deionized water : 100ml

22. Procedure
1. De-wax and hydrate
2. Rinse in deionized water
3. Dip smear in alcian blue for 30 mins
4. Rinse in running water for 5 mins
5. Counterstain with neutral fast red for 10 mins
6. Dehydrate in increasing concentration of alcohol
7. Clear with xylene and mount

23. Results
• Acid mucins, proteoglycans and hyaluronic acid will take blue colour
• Nuclei appear red

24. ALCIAN BLUE – PAS STAINING
• INDICATION
• Combined use of alcian blue and PAS in the same section helps to
differentiate both acidic mucins from neutral mucin in the same
tissue section
• Usually applied in gastrointestinal biopsy sections (goblet cells)

25. Solutions
• Alcian blue, periodic acid and Schiff’s reagent can be prepared as
described in previous sessions

26. Procedure
• De-wax and hydrate
• Rinse in ionized water
• Stain in alcian blue for 30 mins
• Wash in tap water for 2 mins followed by deionized water
• Oxidize in HIO4 for 5-10 mins
• Wash in running tap water for 5 mins
• Add Schiff’s reagent and wash in running tap water
• Counterstain with haematoxylin (*)
• Blue in tap water
• Dehydrate ,clear and mount

27. Results
• Acid mucins = blue
• Glycogen = magenta
• Mixture of acid and neutral mucin = purple

28. Diastase digestion method
• PAS is useful in detecting varied number of mucosubstances i.e
glycogen, mucins and glycoproteins.
• Differentation of both mucins and glycogens using PAS is a
challenge hence the use of diastase method
• This procedure is used for correct identification of
mucosubstances

29. Principle
• -amylase is used to catalyze the hydrolysis of glycosidic
bonds in glycogen and large glycogen molecules thereby
breaking them down to disaccharides making them water
soluble
• The net result is the removal of glycogen from the tissue
before PAS staining

30. Stain preparation
• Malt diastase is frequently used ( contains both - and -maltase)
• Solutions
• Phosphate buffer
• Monobasic sodium phosphate 1.97 g
• Dibasic sodium phosphate 0.28 g
• Distilled water 1000 ml
• This solution may be kept in the refrigerator for several months.
• Diastase solution
• Malt diastase 0.1 g
• Phosphate buffer 100 ml

31. Procedure
• Method
1. Dewax two serial sections in xylene and rehydrate through graded ethanol
to water.
2. Place one slide in the diastase solution for 1 hour at 37°C. The other slide
is an untreated control and may remain in water for 1 hour.
3. Wash both slides in running tap water for 5–10 minutes.
4. Proceed with the PAS technique.

32. Results (with PAS procedure)
• Glycogen should demonstrate bright red/magenta staining in the untreated
slide.
• Glycogen staining should be absent in the diastase treated slide.
• Notes
a. A known positive control should be included to verify the potency
of the enzyme.
b. Commercial batches of diastase or amylase may vary widely in
activity and purity. Contaminating enzymes may digest material other than
glycogen.
c. Alpha-amylase may be used instead of malt diastase.

33. • Questions ?????????