Phosphotungstic Acid Hematoxylin (PTAH) Staining

1. Phosphotungstic Acid
Hematoxylin (PTAH) Staining
Amna sahar
Dmls02161033

2. Objectives
 Purpose
 Clinical significance
 Principle
 Controls
 Fxative
 Section
 Euipments
 Reagents and its preparation
 Procedure
 Result

3. Purpose
 This staining method is used to demonstrate intercellular proteins .
 PTAH is ideal for demonstrating muscles cross striation and fibrin .
 Also used for demonstration of glial fibres.
 There are 3 types of muscles but striation is present in;
 skeletal muscles
 Cardiac muscles

4. Clinical Indications
 Disease caused by gliosis in in central nervous system e.g
Astrocytic tumor
Oligodendroglial tumor
Mixed gliomas etc
 Tumors of skeletal muscles e.g
Sarcomas
Rhabdomyosarcoma etc
 Abnormality in cardiac muscles
Myocardial infarction
 Fibrin deposition in lesions

5. Principle
The amount of phosphotungstic acid in the staining solution is far greater than the amount of
hematoxylin. Phosphotungstic acid binds all the available hematoxylin to form blue lake
pigment. This lake stains muscle cross striations, fibrin, nuclei and other tissue elements
blue. The rest of phosphotungstic acid is thought to stain the components such as collagen
(red-brown)
This stain has been referred to as a polychrome stain because one solution gives two major
colors. The components colored red-brown will lose this color with water or prolonged alcohol
washes, and dehydration of the section following staining therefore must be rapid.

6. CONTROL: Use skeletal or cardiac muscle, tissue containing fibrin for fibrin
staining, and cerebral cortex for glial fibers.
•control slides are intended to be used to verify histological techniques and reagent
reactivity. These slides are to be used for the qualitative purpose of determining
positive or negative results, and are not intended to be used for any quantitative
purpose.
•control slides were produced from human surgical or autopsy tissues under
carefully controlled conditions. Reasonable measures are used to deliver quality
control slides that are as consistent as possible. However, characteristics of quality
control slides may be dissimilar due to variations in the reagents, stains, techniques,
laboratory conditions, and tissue sources used.
FIXATIVE: Zenker's fixative preferred or formalin fixed.
TECHNIQUE: Cut paraffin sections 4 micron for muscle, 6 micron for glial fibers.

7. Controls

8. Equipments:
 Coplin jars
 Microwave oven
 Rinse all glassware in de ionized water

9. Reagents :
 Zenker’s Fluid
 Lugol's Iodine
 5%sodium Thiosulfate
 0.25% Potassium Permanganate
 5% Oxalic Acid
 PTAH Phosphotungstic Acid Hematoxylin Solution

10. Preparation of reagents
 Zenker’s Fluid:
 Distilled water 500.0 ml
 Mercuric chloride 25.0 gm
 Potassium dichromate 12.5 gm
 Sodium sulfate 5.0 gm
 Mix well, label with date and initial. Solution is stable indefinitely.
Note
It contains Mercuric chloride: Severe skin and eye irritant

11.  0.25% Potassium Permanganate:
 Potassium permanganate 0.25 gm
 Distilled water 100.0 ml
 Mix fresh, discard after use
Note
Skin and eye irritant, ingestion will lead to severe gastrointestinal distress.
 5% Oxalic Acid Solution:
 Oxalic acid 5.0 gm
 Distilled water 100.0 ml
 Mix well, label with initial and date. Solution is stable for 1 year.
Note
can cause severe burns of the eyes, skin or mucous membranes. Toxic by inhalation and
ingestion, repeated exposure can cause dermatitis. Corrosive
 Working Solution:
 Right before use, to 50 ml of Zenker’s fluid add 5 ml glacial acetic acid. Discard after
use.
Note
Corrosive poison, discard in labeled glass container

12. Procedure
 Deparaffinize sections thoroughly in three changes of xylene, 3 minutes each.
 Hydrate through two changes each of 100% and 95% ethyl alcohols, 10 dips each. Wash well with
distilled water.
 Fixative Place slides in a plastic Coplin jar containing prepared Zenker Fixative Working Solution and
microwave for 5 minutes at 60°C.
 Wash well in three changes of tap water; rinse in distilled water.
 Remove fixative Lugol's iodine for 5 minutes.
 Wash in tap water.
 Hypo 5% sodium thiosulfate for 1-2 minutes (for glial fibers, bleach out iodine in 95% alcohol in place of
hypo)
 Wash in tap water 10 minutes
 Oxidize in 0.25% potassium permanganate, 5 minutes.
 Wash in tap water.
 Bleach in 5% oxalic acid until white.
 Wash in tap water, rinse in distilled water.
 Staining PTAH stain, overnight, room temperature
 Dehydrate rapidly through two changes each of 95%, 100% alcohol, and two changes of xylene,
coverslip.

13. Result
Cross-striations, fibrin, glial fibers blue
Nuclei blue
Collagen Red-brown
Elastic fibers purplish

14. Phosphotungstic acid-haematoxylin
staining demonstrating contraction band
necrosis in an individual that had a
myocardial infarction (heart attack).

15. Any Question

16. Thank you