Cox Systems Biology

University of Toronto

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Chromogenic Immunohistochemistry (IHC)

Author: Morgan Zych

Contents

Materials

Materials Printable
  • Xylenes
    • Ensure xylenes are handled under sufficient ventilation (fume hood).
  • 100% ethanol
  • 95% ethanol
    • If more 95% ethanol is needed, dilute by adding 41.2mL distilled water to unopened 4L jug of 100% ethanol
  • Distilled water
  • 3% Peroxide Solution
  • Antigen retrieval solution
    • Sodium citrate solution (IHCWorld):
      1. Add 2.94g trisodium citrate to 1000 mL distilled water (milliQ).
      2. Adjust pH to 6.0 or 9.0 (application dependent) using test strips by adding very small amounts of HCl (if too acidic, recover with very tiny amount of NaOH).
      3. Add 0.5mL Tween.
  • Casein blocking buffer
  • Tween 20
  • Unconjugated primary antibodies
  • HRP-conjugated secondary antibodies
  • Humidity chamber
  • PBS (Stock or 10x)
  • DAB kit
    • Carcinogenic. Handle with care & avoid contact.
  • Hematoxylin
  • Bluing buffer (0.1% sodium bicarbonate)
  • Permount slide mounting medium

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Day One

day one Printable
  1. Label all slides w/ pencil.
    • For organoids: do not use slide warmer as it disrupts organoid integrity.
    • For tissues: slide warmer can be used but may cause sections to fall from slides more easily.
  2. Dewax slides by incubate slides in series of 2 buckets of xylenes for 10 minutes each, 2 buckets of 100% ethanol for 5 minutes each, and 2 buckets of 95% ethanol for 5 minutes each.
  3. Incubate slides in bucket of distilled water for 10 minutes
    • While slides incubate in water, make 3% peroxide solution by adding 10mL of 30% stock peroxide to a small bucket and filling bucket up to 100mL line with distilled water.
  4. Incubate slides in bucket of peroxide for 10 minutes
    • Blocks endogenous peroxidase activity to allow specificity of DAB reaction.
    • While slides incubate in peroxide, fill vegetable steamer up to maximum line with distilled water and turn steamer on.
      • Preheat steamer ~10 minutes.
  5. Move slides to a bucket of appropriate antigen retrieval solution*, place bucket in steamer, and steam for one hour.
  6. After antigen retrieval steam, allow slides to cool in freezer, then transfer to a bucket of room temperature distilled water.
    • While slides cool, dilute antibodies in diluent of casein buffer supplemented with 1% Tween.
      • Casein blocks nonspecific antibody binding.
      • Tween increases antibody access to epitopes.
    • Place moistened paper towels inside of humidity chamber and flatten them within grooves to accommodate slides.
  7. Working one slide at a time:
    1. Blot water off of slides at a site far from the sample.
    2. Draw a wide circle (at least 1cm gap between circle + organoids) around sample with hydrophobic pen.
      • Do not press too hard with pen.
      • Ensure each circle is complete.
    3. Allow pen to dry very briefly then transfer slide into humidity chamber.
    4. Add 200mL of diluted antibody or control solution (diluent only) to each slide.
      1. If necessary, gently disperse antibody solution with pipette tip just contacting the solution itself, not touching the sample/slide!
  8. Place lid on humidity chamber and carefully move the chamber to the +4oC fridge for overnight incubation of primary antibodies.

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Day Two

day two Printable
  1. Prepare three 250mL wash buckets by adding 25mL of 10X PBS to each bucket, then topping each bucket up to the 250mL line with distilled water.
    • Add 2.5mL Tween (1%) as well to the middle bucket in the series of three washes.
  2. Take slides out of the humidity chamber and check to make sure none of the slides became dry overnight.
    • If this occurred, make a note of which samples this happened to and consider removing from analysis.
  3. Move slides to a slide rack while holding the rack above the first wash bucket so that the liquid on the slides is caught by the bucket. Place in this bucket for 5 minutes.
  4. Wash in second (Tween-containing) and then third bucket for 5 minutes each.
  5. Set a timer for 30 minutes. Blot the first slide and apply minimum necessary amount of drops of species-appropriate secondary antibody onto it. Start the timer and apply secondary antibody to each subsequent slide in regular intervals (30 seconds, 1 minute, whatever works) keeping the slides in a line in the order they received secondary antibody.
    • Incubate at room temperature.
    • Disperse antibodies with pipette tip if necessary.
    • While secondary antibodies incubate, prepare all materials needed to finish slide prep:
      • Make DAB solution by calculating the volume needed to apply 200mL per slide, then adding 30mL DAB enzyme for every 1000mL of DAB diluent needed. Invert tube repeatedly to mix
      • Fill a 100mL bucket with tap water (NOT distilled this time) and place slide rack into tap water so it is ready.
      • Place kimwipes or white piece of paper near this bucket for checking DAB development.
      • Empty a wash bucket, rinse, and fill it to the 250mL line with tap water. Place beside hematoxylin in fume hood.
      • Fill 2 buckets each with 95% ethanol, 100% ethanol, and xylenes.
  6. Transfer slides into rack and into the first wash bucket at the same time interval as you applied secondary antibody.
    • This ensures that each slide incubates with secondary antibody for a full 30 minutes.
  7. When each slide has been placed in the rack in the first wash bucket, perform all 3 washes for 5 minutes each. 
  8. Reset timer. Choose which slide should receive DAB first; if optimizing, choose the most concentrated antibody prep. Do not use control as first slide. Add 200mL DAB solution to this slide and start the timer.
    • As with secondary antibody application, add DAB solution to each subsequent slide at a regular time interval
    • Check DAB development as you go! Amount of time needed with DAB is variable but usually within 2-7 minutes.
  9. When DAB development is sufficient, place slides into rack in the tap water bucket. Tap water stops the DAB reaction.
    • Stop DAB reaction after each slide has incubated for the same amount of time by adding them to the bucket at the same time interval DAB was applied.
    • Consider working with a partner if processing large numbers of slides.
  10. When all slides have been placed into the tap water bucket, transfer loaded rack into the hematoxylin bucket in the fume hood and incubate for two minutes.
  11. Move slides into bucket of water. Bring this bucket to the sink and replace water + wash slides until water is clear.
    • Do not let slides come into contact with the running tap.
  12. Dip rack of slides into bucket of bluing buffer 7 times.
  13. Clear slides by dipping them 15 times each in 2 buckets of 95% ethanol, 2 buckets of 100% ethanol, and 2 buckets of xylenes in that order. Leave slide rack in final xylene bucket while mounting slides
  14. With small pestle put Permount on the slide encircling the sample. Squish slide cover on and press to disperse Permount on top of sample and to push any bubbles away.
    • Keep in the fume hood
  15. Wait at least 2 hours, then gently clean slides with kimwipe dipped in xylene.
  16. Wait a few hours more until all xylenes have evaporated and mounting media is completely hardened before viewing/imaging

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Last updated: 02/2025