DAVAO DOCTORS COLLEGE

MEDICAL LABORATORY SCIENCE DEPARTMENT

STUDENT NOTES: HPCT

H&E STAINING TECHNIQUE

H&E Method for Frozen Sections
2 Major Group ▪ Hematoxylin-Eosin Method
- applicable in all organs and disease model
▪ Histological Staining ▪ Thionine Method
- Direct interaction with a dye or staining solution - brain sections and visualizing macroscopic
- Produce coloration of active tissue component lesions
- Ex. Microanatomic Stain, Bacterial Stain, Tissue ▪ Polychrome Methylene Blue Method
Stain - paraffin sections fixed in formaldehyde
▪ Histochemistry ▪ Alcoholic Pinacyanol Method
- Chemical reaction - supravital staining of Mitochondria
- Permit microscopic localization of specific tissue - color sensitization in photography
substance
- Ex. Perl’s Prussian Blue, PAS Frozen Section Technique for Rapid Diagnosis
1. Fix a selected portion of tissue by dropping it into
Hematoxylin-Eosin Staining boiling 10% Formalin for 2 minutes.
▪ Most common method 2. Wash rapidly in tap water.
▪ Regressive staining 3. Mount onto blockholder and freeze.
- Overstaining the Nuclei 4. Cut sections at 10 micra.
- Removal of superfluous 5. Mount section. (Albuminized slides)
- Acid differentiation 6. Rinse rapidly in water.
▪ Fixed w/ Mercuric Chloride 7. Harris Hematoxylin for 30-45 seconds
- Increase Hematoxylin 8. Rinse in tap water.
- Reduce Eosin 9. Blue in Ammonia Water for 5 sec.
▪ Prolonged Staining 10. Rinse in tap water.
- Chromium and Osmium fixed tissues 11. Place in 5% Aqueous Eosin
- long Acid Decalcification 12. Rinse in tap water.
- Prolonged storage in Acid Formalin or 70% 13. Dehydrate, clear and mount.
Alcohol
▪ Not demonstrated RESULT
- Neuroglia fibers, axon, nerve endings, reticulum, Nuclei: BLUE
golgi bodies Cytoplasm: PINK
and mitochondria Erythrocytes:BRIGHTRED
All other eosinophilic structures: RED, PINK. ORANGE
The staining procedure for H&E follows a basic protocol:
▪ Dewaxing HEIDENHAIN’S IRON HEMATOXYLIN METHOD
▪ Dehydration ▪ Solution 1
▪ Hematoxylin Iron Alum: 2.5 gm.
▪ Differentiation Distilled Water: 100 ml
▪ Bluing ▪ Solution 2
▪ Eosin Hematoxylin: 0.5 gm
▪ Dehydration Ethyl Alcohol 95: 10 ml
▪ Clearing Distilled Water: 90 ml
▪ Cover-slipping
PROCEDURE
1. Sections to water.
2. Mordant in solution 1 for 3 hours or longer.
3. Rinse in distilled water.
4. Stain in solution 2 for another 3 hours or longer.
5. Rinse water.
6. Differentiate in solution 1.
7. Wash in running water for 5-10 mins.
8. Counterstain as required.
9. Dehydrate, clear, and mount

RESULT
- BLACK
(nuclei, cytoplasmic inclusions and muscle striations)

CELESTINE BLUE-HAEMALUM SEQUENCE STAINING

▪ Solution 1
Iron Alum: 25 gm.
Celestine Blue (CI 900): 1.25 gm.
Glycerol 35 ml.
Distilled Water: 250 ml
▪ Solution 2
Mayer’s Acid Alum Hematoxylin

PROCEDURE
1. Sections to water.
2. Stain in Solution 1 for 10-20 minutes
3. Rinse in water.
4. Stain in Mayer’s Acid Alum Hematoxylin for 5-10
mins.
5. Rinse water.
6. Blue in running tap water
7. Counterstain as required.
8. Dehydrate, clear, and mount

RESULT: BLUE (Cell Nuclei)

MALLORY’S PHLOXINE METHYLENE BLUE STAIN

▪ Original Name: Eosin-Methylene Blue Method
▪ Produces sharp nuclear stain
▪ Marked differentiation
▪ Should be fixed in Zenker’s fluid