TITLE: Immunohistochemistry on free-floating and paraffin-embedded tissue sections
AUTHORS: Ching-Chieh Chou, Judith Frydman

[DESCRIPTION]
This protocol is used for free-floating frozen (30-50 microns) and paraffin-embedded (10 microns) tissue sections.

[STEPS]
SECTION: Materials
1. Free-floating
PBS
0.3% Triton-X-100 in PBS 1x, stored at 4°C
Normal Donkey Serum (NDS), stored at -20°C
Blocking Buffer: 10% NDS and 0.03% Triton-X-100 in PBS 1x. Diluted from 100% NDS (stored at -20°C) and 0.3% Triton-X-100. Filtered with 0.22 µM filter.
Primary Antibody
Secondary Antibody
Hoechst dye
Mounting medium

Paraffin-embedded
Deparaffinization station
Slide rack
Glass slide holder and tray
Humidity and slide incubation chamber
Xylene
Ethanol
Deionized water (diH2O)
PBS
Citrate buffer, pH 6.0, 10x
0.3% Triton-X-100 in PBS 1x, stored at 4°C
Normal Donkey Serum (NDS), stored at -20°C
Blocking Buffer: 10% NDS and 0.03% Triton-X-100 in PBS 1x. Diluted from 100% NDS (stored at -20°C) and 0.3% Triton-X-100. Filtered with 0.22 µM filter.
Hydrophobic Barrier Pap Pen
Primary Antibody
Secondary Antibody
Hoechst dye
Mounting medium
SECTION: Procedure for free-floating tissue staining
2. Collect free-floating frozen tissue sections and process in a 24-well culture plate.
SECTION: Procedure for free-floating tissue staining
2.1. Day 1: Wash the tissue for 3 X 5 min in PBS 1x.
SECTION: Procedure for free-floating tissue staining
2.2. Permeabilize the tissue in 0.3% Triton-X-100 in PBS 1x for 20 min at room temperature on a rocking shaker.
SECTION: Procedure for free-floating tissue staining
2.3. Wash the tissue for 3 X 5 min in PBS 1x.
SECTION: Procedure for free-floating tissue staining
2.4. Block the tissue in Blocking Buffer for 1 hr at room temperature on the rocking shaker.
SECTION: Procedure for free-floating tissue staining
2.5. Briefly rinse the tissue in PBS 1x once.
SECTION: Procedure for free-floating tissue staining
2.6. Prepare Primary Antibody in Blocking Buffer.
SECTION: Procedure for free-floating tissue staining
2.7. Incubate the tissue with Primary Antibody and transfer the plate to a refrigerator for overnight at 4°C.
SECTION: Procedure for free-floating tissue staining
2.8. Day 2: Wash the tissue for 3 X 5 min in PBS 1x.
SECTION: Procedure for free-floating tissue staining
2.9. Prepare Secondary Antibody in filtered 10% NDS in PBS 1x.
SECTION: Procedure for free-floating tissue staining
2.10. Incubate the tissue with Secondary Antibody for 1 hr at room temperature on the rocking shaker in the dark.
 

SECTION: Procedure for free-floating tissue staining
2.11. Wash the tissue for 2 X 5 min in PBS 1x.
SECTION: Procedure for free-floating tissue staining
2.12. Incubate the tissue in Hoechst (1:2000) in PBS 1x for 10 min at room temperature on the rocking shaker.
SECTION: Procedure for free-floating tissue staining
2.13. Rinse the tissue for 2 X 5 min in PBS 1x.
SECTION: Procedure for free-floating tissue staining
2.14. Transfer the tissue to a slide and remove solution around the tissue by Kimwipes.
SECTION: Procedure for free-floating tissue staining
2.15. Add mounting medium and put on a coverslip starting with top slowly to avoid generating any bubbles.
SECTION: Procedure for free-floating tissue staining
2.16. Gently move the coverslip and let the mounting medium cover the edge of the slide. Let it dry overnight in the dark.
SECTION: Procedure for free-floating tissue staining
2.17. Day 3: Acquire imaging by a microscope.
SECTION: Procedure for paraffin-embedded tissue staining
3. Process paraffin-embedded tissue sections on glass slides.
SECTION: Procedure for paraffin-embedded tissue staining
3.1. Day 1: Place the slides in a slide rack and rehydrate sections by sequential washes in a deparaffinization station:
Xylene: 2 x 5 min
100% Ethanol: 2 x 10 min
95% Ethanol: 10 min
70% Ethanol: 10 min
50% Ethanol: 10 min
diH2O: 2 x 5 min
SECTION: Procedure for paraffin-embedded tissue staining
3.2. For antigen retrieval, dilute Citrate buffer in water to make 1x solution.
SECTION: Procedure for paraffin-embedded tissue staining
3.3. Fill a beaker with Citrate buffer 1x and place the beaker on a hot plate covered with foil. Boil the buffer to 100°C. 
SECTION: Procedure for paraffin-embedded tissue staining
3.4. After the final step of rehydration, transfer the slides from the slide rack to a glass slide holder. Place the holder in the beaker and heat the slides for 20 min.
 

SECTION: Procedure for paraffin-embedded tissue staining
3.5. Take off the beaker from the hot plate and let it cool for 20 min.
SECTION: Procedure for paraffin-embedded tissue staining
3.6. Add PBS 1x to a glass tray. Put the glass slide holder in the tray and wash for 3 X 5 min on the rocking shaker.
SECTION: Procedure for paraffin-embedded tissue staining
3.7. After PBS wash, carefully remove all the solution around the section with Kimwipes and draw a circle around the tissue with PAP pen a few times.
SECTION: Procedure for paraffin-embedded tissue staining
3.8. Add Blocking Buffer (~200 µL) to cover the tissue without going over the PAP pen repellent border. Incubate for 2 hr at room temperature.
SECTION: Procedure for paraffin-embedded tissue staining
3.9. Prepare Primary Antibody in Blocking Buffer.
SECTION: Procedure for paraffin-embedded tissue staining
3.10. Remove all the Blocking Buffer around the tissue with Kimwipes.
SECTION: Procedure for paraffin-embedded tissue staining
3.11. Transfer the slides to a humidity and slide incubation chamber. Add water to bottom of incubation chamber.
SECTION: Procedure for paraffin-embedded tissue staining
3.12. Incubate the tissue with Primary Antibody (~200 µL) and transfer the chamber to cold room for overnight at 4°C.
SECTION: Procedure for paraffin-embedded tissue staining
3.13. Day 2: Knock the slides gently on Kimwipes and dry around the tissue.
SECTION: Procedure for paraffin-embedded tissue staining
3.14. Stagger slides in the glace slide tray and wash with PBS 1x for 3 X 5 min on the rocking shaker.
SECTION: Procedure for paraffin-embedded tissue staining
3.15. Prepare Secondary Antibody in 0.03% Triton-X-100 in PBS 1x.
SECTION: Procedure for paraffin-embedded tissue staining
3.16. After PBS wash, remove all the solution around the tissue with Kimwipes.
SECTION: Procedure for paraffin-embedded tissue staining
3.17. Transfer the slides to the humidity and slide incubation chamber. Add water to bottom of incubation chamber.
SECTION: Procedure for paraffin-embedded tissue staining
3.18. Incubate the tissue with Secondary Antibody (~200 µL) for 1 hr at room temperature in the dark.
 

SECTION: Procedure for paraffin-embedded tissue staining
3.19. Gently wipe off the solution around the tissue. Stagger slides in the glace slide tray and wash with PBS 1x for 2 X 5 min on the rocking shaker, covered with foil.
SECTION: Procedure for paraffin-embedded tissue staining
3.20. Remove solution around the tissue. Transfer the slides to the humidity and slide incubation chamber.
SECTION: Procedure for paraffin-embedded tissue staining
3.21. Incubate with Hoechst dye at 1:2000 dilution in PBS 1x for 10 min.
SECTION: Procedure for paraffin-embedded tissue staining
3.22. Place the slides in the glace slide tray and rinse with PBS 1x for 2 X 5 min on the rocking shaker.
SECTION: Procedure for paraffin-embedded tissue staining
3.23. After the final wash, gently tap on the slides and remove all the solution around the tissue. Dry the slides onvernight in the dark.
SECTION: Procedure for paraffin-embedded tissue staining
3.24. Day 3: Add mounting medium on the coverslip. Place the slides on the coverslip slowly to avoid generating any bubbles. 
SECTION: Procedure for paraffin-embedded tissue staining
3.25. Flip the slides with coverslip on the top. Gently move the coverslip and let the mounting medium cover the edge of the slide. 
SECTION: Procedure for paraffin-embedded tissue staining
3.26. Dry the slide overnight in the dark.
SECTION: Procedure for paraffin-embedded tissue staining
3.27. Day 4: Acquire imaging by a microscope.