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H&E Staining, Masson Trichrome Staining, and Immunohistochemistry of Kidney Tissues
Published: Jun 23, 2021 Views: 941
Original research article
The authors used this protocol in:
Jan 1, 2020
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Hematoxylin and Eosin (H&E) staining
1. Serial tissue sections (4 μm) of kidneys were sliced from paraffin-embedded, formalin-fixed kidneys
2. Remove the wax with xylene two times for 5 minutes
3. Hydrate the section with different concentration of Ethanol ,(100% and 95%) two times for 5 minutes, 75% Ethanol for 5 minutes, 50% Ethanol for 5 minutes.
4. Rinse in running tap MQ water for 5 minutes.
5. Rinse in 1x PBS.
6. Stain nuclei with hematoxylin for 2 minutes.
7. Rinse in running tap MQ water.
8. Cover slide with 0.1% ammonia water and rinse in running tap MQ water for 5 minutes to fade.
9. Stain the cytoplasm with eosin for 5 seconds.
10. Dehydrate the slide with different concentration of Ethanol.
11. Clear the slide with xylene two times for 5 minutes.
12. Mount the slide with mounting medium and cover the glass coverslip with a 45 degree angle to avoid air bubbles.
13. After the mounting medium has solidified, the staining is completed.
Masson's trichrome staining
1. Incubate the slides in oven for 10 minutes.
2. Remove the wax with xylene two times for 5 minutes
3. Hhydrate the section with different concentration of Ethanol (100% and 95%) two times for 5 minutes, 75% Ethanol for 5 minutes, 50% Ethanol for 5 minutes.
4. Rinse in running tap MQ water for 5 minutes.
5. Immerse the slide in the preheated Bouin’s solution for 1 hour at 60oC in an oven.
6. Rinse in running tap water for 5 minutes to remove the yellow color.
7. Stain with Weigert's Iron Hematoxylin solution for 5 minutes.
8. Rinse in running tap water for 5 minutes and then wash slide in 70 rpm MQ water.
9. Stain with Biebrich Scarlet Acid Fuchsin solution (Red) for 10 minutes.
10. Rinse in running tap MQ water.
11. Immerse the slide in Working Phosphotungstic-Phosphomolybdic Acid solution for 15 minutes.
12. Stain with Aniline Blue solution (Blue) for 30 minutes.
13. Rinse in running tap MQ water.
14. Immerse in 1% acetic acid solution for 3 minutes.
15. Rinse in running tap MQ water.
16. Dehydrate the slide with different concentration of Ethanol.
17. Clear the slide with xylene two times for 5 minutes
18. Mount the slide with mounting medium and cover the glass coverslip with a 45 degree angle to avoid air bubbles.
19. After the mounting medium has solidified, the staining is completed.
20. Staining showed collagen fibers in blue and cytoplasm in red color.
Immunohistochemistry (IHC)
Deparaffinization and hydrate
1. Incubate the slides in oven for 10 minutes.
2. Remove the wax with xylene two times for 5 minutes
3. Hhydrate the section with different concentration of Ethanol (100% and 95%) two times for 5 minutes, 75% Ethanol for 5 minutes, 50% Ethanol for 5 minutes.
4. Rinse in running tap MQ water for 5 minutes.
5. Immerse in 1x PBS for 3 minutes.
Antigen retrieval
- Immerse in boiled 0.01M Sodium citrate solution with pH 6.0 two times for 7 minutes.
- Put the slide into ice to cool down.
- Immerse in 1x PBS two times for 3 minutes (100 rpm).
- Immerse in 3% H2O2/Methanol for 10 minutes to block enzyme activity (70 rpm).
- Immerse in 1x PBS two times for 3 minutes (100 rpm).
Immunostain
1. Use STARR TREK Universal HRP detection kit (Biocare medical, Concord, CA, United States).
2. Immerse in background eraser for 15 minutes (Light blue).
3. Wipe off excess liquid.
4. Apply 50 µl diluted primary antibody (e.g. 1% BSA in PBS) to the sections on the slide and incubate in room temperature for 1 hour.
5. Immerse in 1x PBS two times for 5 minutes (100 rpm).
6. Immerse in trekkie universal link for 20 minutes (Yellow).
7. Immerse in 1x PBS two times for 2 minutes (100 rpm).
8. Apply secondary antibody TrekAvidin-HRP to the sections on the slide and incubate in room temperature for 20 minutes.
9. Immerse in 1x PBS two times for 2 minutes (100 rpm).
10. Apply 20µl diluted DAB(1ml buffered substrate+1 drop DAB) substrate solution/per section to the sections to reveal the color and observe the color with a microscope.
11. Immerse in TBST to stop coloring.
12. Stain with hematoxylin to compared with the immunostain.
13. Dehydrate the slide with different concentration of Ethanol.
17. Clear the slide with xylene two times for 5 minutes
18. Mount the slide with mounting medium and cover the glass coverslip with a 45 degree angle to avoid air bubbles.
19. After the mounting medium has solidified, the staining is completed.
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