Immunohistochemistry for IBA-1

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  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)


  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