Hematoxylin and Eosin Stain
(H&E)
TECHNIQUE: Formalin fixed, paraffin tissue sections
REAGENTS:
Mayer’s Hematoxylin (Source Medical, catalog# 9235360)
0.5% Acid Alcohol
70% Ethanol ----------------------------------------------------------------------995ml
Hydrochloric Acid, (36.5% - 38%) ------------------------------------------- 5ml
1X PBS (pH 7.2-7.3)
Alcoholic-Eosin
1.0% Eosin Y
Eosin Y (CAS# 17372-87-1) -------------------------------------------------- 1.0 g
Distilled water ------------------------------------------------------------------ 100 ml
1.0% Phloxine B
Phloxine B (CAS# 18472-87-2) ---------------------------------------------- 0.5 g
Distilled water ------------------------------------------------------------------ 50.0 ml
Working Alcoholic-Eosin Solution
1.0% Eosin Y ------------------------------------------------------------------- 50.0 ml
1.0% Phloxine B --------------------------------------------------------------- 5.0 ml
95% Ethanol -------------------------------------------------------------------- 390.0 ml
Acetic acid, glacial ------------------------------------------------------------- 4.0 ml
11/2018
Hematoxylin and Eosin Stain
(H&E)
5 micron Paraffin Sections
PROCEDURE:
1. Deparaffinize and rehydrate slides to distilled water
2. Stain in Mayers Hematoxylin for 1 minute
3. Wash with 4-5 changes of Tap water or until blue stops coming off slides
4. Blue nuclei in 1X PBS for 1 minute
5. Wash with 3 changes of distilled water
6. Counterstain in Alcoholic-Eosin for 1 minute (DO NOT RINSE)
7. Dehydrate through 3 changes of 95% EtOH and 2 changes of 100% EtOH 1 minute each
8. Clear in 3 changes of Xylene 1 minute each change
9. Mount and coverslip
*Do NOT rinse with water after Alcoholic-Eosin! Go directly into dehydration of slides.
RESULTS:
Nuclei ------------------------------------------ blue
Cytoplasm ------------------------------------ pink
Erythrocytes --------------------------------- bright red
All other eosinophilic structures --------- red, pink, or orange
8/2017
Hematoxylin and Eosin Stain For Fresh Frozen Section
(H&E)
PROCEDURE:
1. Take slides immediately from freezer and place in cold fixative (10% Neutral Buffered Formalin or 4% PFA) for
10 minutes
2. Rinse slides in 1X PBS, 2 times for 3 minutes each to remove OCT or other tissue embedding compound
3. Rinse in a gentle stream of tap water for 1 minute
4. Stain in Mayers Hematoxylin for 30 seconds
5. Wash in a gentle stream of tap water for 1 minute
6. Blue nuclei in 1X PBS for 20 seconds
7. Wash in a gentle stream of tap water for 1 minute
8. Dip in 70% EtOH for 30 seconds
9. Dip in 95% EtOH for 30 seconds
10. Counterstain in Alcoholic-Eosin for 30 seconds
11. Dehydrate through 2 changes of 95% EtOH for 15 seconds
12. Dehydrate through 3changes of 100% EtOH for 15 seconds
13. Clear through 3changes of Xylene for 1 minute each and coverslip
RESULTS:
Nuclei ------------------------------------------ blue
Cytoplasm ------------------------------------ pink
Erythrocytes --------------------------------- bright red
All other eosinophilic structures --------- red, pink, or orange
8/2017
Hematoxylin and Eosin Stain For Frozen Section
(H&E)
PROCEDURE:
1. Rinse slides in 1X PBS for 3 minutes
2. Rinse in a gentle stream of tap water until OCT is washed off for 3 minutes
3. Stain in Mayers Hematoxylin for 30 seconds
4. Wash in a gentle stream of tap water for 1 minute
5. Blue nuclei in 1X PBS for 20 seconds
6. Wash in a gentle stream of tap water for 1 minute
7. Dip in 70% EtOH for 30 seconds
8. Dip in 95% EtOH for 30 seconds
9. Counterstain in Alcoholic-Eosin for 30 seconds
10. Dehydrate through 2 changes of 95% EtOH for 15 seconds
11. Dehydrate through 3changes of 100% EtOH for 15 seconds
12. Clear through 3changes of Xylene for 1 minute each and coverslip
RESULTS:
Nuclei ------------------------------------------ blue
Cytoplasm ------------------------------------ pink
Erythrocytes --------------------------------- bright red
All other eosinophilic structures --------- red, pink, or orange
8/2017
Hematoxylin and Eosin Stain for soft tissue
(H&E)
4 micron Paraffin Sections
PROCEDURE:
1. Deparaffinize and rehydrate slides to distilled water
2. Stain in Mayers Hematoxylin for 1 minute
3. Wash with 4-5 changes of Tap water or until blue stops coming off slides
4. Decolorize with 3 dips in 0.5% acid alcohol
5. Wash in 3 changes of distilled water
6. Blue nuclei in 1X PBS for 1 minute
7. Wash with 3 changes of distilled water
8. Counterstain in Alcoholic-Eosin for 30 seconds (DO NOT RINSE)
9. Dehydrate through 3 changes of 95% EtOH and 2 changes of 100% EtOH 1 minute each
10. Clear in 3 changes of Xylene 1 minute each change
11. Mount and coverslip
*Do NOT rinse with water after Alcoholic-Eosin! Go directly into dehydration of slides.
RESULTS:
Nuclei ------------------------------------------ blue
Cytoplasm ------------------------------------ pink
Erythrocytes --------------------------------- bright red
All other eosinophilic structures --------- red, pink, or orange
11/2018
