Immunohistochemistry and Immunocytochemistry: Tissue Imaging via Light Microscopy

1. Preparation of Cells for Immunocytochemistry

1. Seed cells of interest onto chambered slides or chambered coverslips by adding 0.5 mL of cell suspension to the wells of a 24 well culture plate.
   Note: Some cells may require growth on treated coverslips, such as coverslips treated with poly-lysine. Optimal treatment conditions should be determined by the user depending on the cell type being used.
2. Place the plate into a humidified CO₂ incubator and allow the cells to grow at 37°C until 50-70% confluent.
3. Once the cells reach optimal confluency, remove the culture media from each well and then, fix the cells by incubating them in 0.5 mL of 4% PFA (diluted in 1X PBS) and incubate for 20 min at room temperature.
4. Remove the fixative and wash the wells three times with 1 mL of 1X PBS.
5. Next, permeabilize the cells by adding 0.5 mL of 0.1% Triton-X-100 in 1X PBS to each well and incubate for 15 min at room temperature.
6. Aspirate off the permeabilization buffer and wash the wells thrice with 1 mL of 1X PBS.
7. The cells on coverslips are now fixed and permeabilized. Proceed to the staining procedure demonstrated for the following immunohistochemistry example- with the exception that the incubations should be performed within the wells of the 24 well plate rather than directly on a tissue section slide.

2. Preparation of Formalin-Fixed, Paraffin-Embedded Sections for Staining

1. Obtain prepared formalin-fixed, paraffin embedded tissue sections.

Deparaffinization

2. Immerse the slides in 100% xylene 2 times for 5 min each.

Rehydration

3. Immerse the slides in 100% ethanol 2 times for 3 min each.
4. Immerse the slides in 95% ethanol for 3 min.
5. Immerse the slides in 70% ethanol for 3 min.
6. Immerse the slides in 50% ethanol for 3 min.

Blocking Endogenous Peroxidase Activity

7. Incubate the slides in 100 mL of 0.3% H₂O₂ for 30 min at room temperature.
8. Wash the slides with 1X PBS 2 times for 5 min each.

Antigen Retrieval

9. Immerse the slides in IHC citrate buffer (pH 6) and boil them for 20 min.
10. Tissue slides are now ready for staining.

3. Preparation of Fresh-Frozen, OTC-Embedded Sections for Staining

1. Place 5 mm of fresh isolated tissue into a mold and add OCT until the section is completely covered.
2. Slowly submerge the tissue block into liquid nitrogen until completely frozen. The sample can now be stored at -80°C for up to 1 year.
3. Once ready for sectioning, transfer the frozen tissue block to a cryostat and allow the entire setup to come to -20°C.
4. Cut 5-10 µm thick tissue sections using a cryostat and use brush to place sections directly onto IHC compatible glass slides.
5. Allow slides to dry overnight at room temperature. The slides can also be stored at -80°C.
6. Immerse the slides in 250 mL of 4% PFA for 15 min at room temperature to fix the slides prior to staining. Optimal fixation method should be determined by user.
7. Immerse the slides in 250 mL of 1X PBS 2 times for 5 min each.
8. Immerse the slides in 250 mL of 0.3% H₂O₂ for 30 min at room temperature in order to block any endogenous peroxidase activity.
9. Immerse the slides in 250 mL of 1X PBS 2 times for 5 min each.
10. The slides are now ready for staining.

4. Staining

1. Circle the tissue with a hydrophobic barrier using a barrier pen.

Blocking

2. Using a pipette, place 100 µL of blocking buffer (horse serum diluted in 1X PBS) - over the section for 1 hour at room temperature.
3. Remove blocking buffer using a pipette.

Primary Antibody Incubation

4. Incubate the encircled tissue with 100 µL diluted primary antibody solution (mouse anti-human cyclin D1 diluted 1:100 in blocking buffer) for 30 min at room temperature.
5. Drain the primary antibody off each slide and wash the slides with 1X PBS 2 times for 5 min each.

Secondary Antibody Incubation

6. Incubate the sample with 100 µL of diluted biotinylated secondary antibody (biotinylated horse anti-mouse IgG diluted 1:200) for 30 min at room temperature.
7. Remove the secondary antibody by draining off the sections and wash with 1X PBS 2 times for 5 min each.

Color Development

8. Visualization using HRP: Add 100 µL of avidin-biotin complex (ABC) reagent and incubate the sections in dark in for 30 min at room temperature
   Note: Fluorescently tagged antibodies can also be used and visualized using an appropriate microscope.
9. Wash the slides with 1X PBS 2 times for 5 min each.
10. Develop the slides by incubating the sections in 100 µL of DAB for up to 5 min.
11. Stop the development by adding distilled water (dH₂O) for 5 min at room temperature.

Counterstaining (if desired)

12. Immerse the slides briefly in Harris Hematoxylin Solution (or 0.5% methyl green in 0.1M sodium acetate (pH 4.2)) for 10 min.
13. Rinse off the counterstain by washing slides in dH₂O two times for 5 min each.

Dehydration

14. Immerse the slides in 95% ethanol 2 times for 5 min each.
15. Immerse the slides in 100% ethanol 2 times for 5 min each.
16. Immerse the slides in 100% xylene 2 times for 5 min each.
17. Blot the slides with a paper towel.

Mounting and coverslip application

18. Add a drop of mounting media such as Organo-Limonene Mount to the slides and place a coverslip over the sections.

Microscopic analysis

19. Observe the stained sections under an appropriate microscope for analysis. Here a standard light microscope was used for observation and a mounted digital camera was used for imaging.