Immunohistochemistry for IBA-1

Deparaffinizing

1. Bake slides for an hour at 60°C
2. Deparaffinize tissue in four changes of xylene -X1 (5 min), X2 (3 min), X3 (3 min), X4 (3 min)
3. Rehydrate slides by placing in 100% EtOH (2x) (2 min each)
4. Place in 95% EtOH (3x) (2 min each)
5. Place in 70% EtOH (2 min)
6. Place in distilled water (2 min)

From this point on slides are not allowed to dry

Antigen retrieval - use only plastic coplin jars / slide racks

1. Preheat citrate buffer (9 ml of 0.1M citric acid + 41 ml of 0.1M sodium citrate dihydrate, bring final volume to 500 ml with distilled water and bring pH to 6 with 5N NaOH)
2. Place slides in citrate buffer and put the coplin jar in the pressure cooker for 10 min
3. Cool slides to room temp → to speed up the process you can place the coplin jar in a dish and run cool water beside it
4. Rinse slides in distilled water (2x)

Staining

1. Create hydrophobic barriers using IHC pen
2. Wash the slides once in wash buffer (0.05% Tween-20, 1x PBS) for 5 min - Approximately 300 ul per slide
3. Block for 1 hour at room temperature with blocking buffer (5% serum in wash buffer)
4. Remove the blocking buffer and add the primary antibody → dilute primary antibody in wash buffer with 2.5% serum (1:500). Incubate overnight at 4°C
5. Remove antibody and wash slides 3x for 5 min with wash buffer
6. Add secondary antibody (dilute in wash buffer 1:200) and incubate at room temperature for 1h
7. Remove secondary antibody and wash 3x for 5 min
8. Make ABC (2 drops reagent A and 2 drops reagent B in 5 ml wash buffer) → has to sit for a least 30 min
9. Wash 3x for 5 min
10. Add DAB and urea tablet to 15 mL of DD water
11. Add DAB to slides and wait ~ 1-5 min
12. Dispose of all tips in special waste and add bleach to DAB and DAB waste after finishing
13. Rinse slides in tap water
14. (optional) - for hemotoxylin staining, dip slides for 10 minutes in hemotoxylin and rinse in tap water for 5 minutes

Dehydration and Coverslip

1. From tap water, place slides in line distilled water for 2 min
2. 70% EtOH for 5 dips
3. 95% EtOH (4x for 2 min each)
4. 100 EtOH (2x for 2 min each)
5. 100 EtOH (2x for 3 min each)
6. Xylene (4x for 2 min each)
7. Coverslip with Permount Solution