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. Author manuscript; available in PMC: 2017 Apr 1.
Published in final edited form as: Differentiation. 2015 Nov 3;91(4-5):139–151. doi: 10.1016/j.diff.2015.10.013

Table 6.

General protocol for Immunohistochemistry.

  • Day 1 – 3 hour protocol:

  • Depariffinization

    • 1

      Submerge slides in Histoclear two times for 10 minutes.

  • Rehydration

    • 2

      Submerge slides in a series of graded ethanol washes.

      1. 100% ethanol for 10 minutes.

      2. 95% ethanol for 10 minutes.

      3. 70% ethanol for 10 minutes.

    • 3

      To block endogenous peroxidase place slides in 0.3% H2O2 in MetOH @ RT for 30 minutes.

    • 4

      Wash in running distilled water for 5 minutes.

  • Antigen Retrieval

    • 5

      Dilute 10x Antigen Retrieval Solution to 1x in distilled water.

    • 6

      Pre-heat 1x Antigen Retrieval Solution for 5 min in microwave (100% power).

    • 7

      Place the tissues into a container with enough 1X target retrieval solution to completely cover slides.

    • 8

      Cook slides in microwave for 20 minutes at 30% power – watch for evaporation.

    • 9

      Cool to room temperature for 20 minutes.

    • 10

      Rinse slides in 1X PBS for 5 minutes.

    • 11

      Incubate in 2.5% horse serum (diluted in 1X PBS) for 60 minutes at room temperature.

    • 12

      Dilute Primary antibody in 1X PBS + 2.5% horse serum (prepare ≈75 μL per slide).

    • 13

      Incubate slides in primary antibody overnight at 4°C.

  • Day 2 – 5.5 hour protocol:

    • 14

      Wash in 1x PBS two times for 30 minutes at 4°C.

    • 15

      Dilute secondary antibody in 1x PBS + 2.5% horse serum (prepare ≈75 μL per slide).

    • 16

      Incubate slides in secondary antibody at room temperature for 60 minutes.

    • 17

      Wash in 1x PBS two times for 30 minutes at 4°C.

      *Note: after first 30 minutes take out and prepare Vectastain ABC reagent.

    • 18

      Incubate with Vectastain ABC reagent at room temperature for 60 minutes.

    • 19

      Wash in 1x PBS two times for 30 minutes a 4°C.

    • 20

      Develop with DAB at room temperature until stained.

    • 21

      Wash in running tap water for 10 minutes.

      *Note: tap water is alkaline and allows for better counterstain.

    • 22

      Incubate in hematoxylin at room temperature for 1–2 minutes (time varies depending on time of hematoxylin used).

    • 23

      Wash in running tap water for 2–3 minutes.

    • 24

      Dip 3 times in 1% acid alcohol

    • 25

      Wash in running tap water for 2 min

    • 26

      Dip 10 times (or until blue) in 0.3% ammonia water.

    • 27

      Wash in running tap water for 1 minute.

    • 28

      Dehydrate in 70% EtOH – 2 × 10 dips, 95% EtOH – 2 × 10 dips, 100% EtOH – 2 × 10 dips and Histoclear 3 × 20 dips.

    • 29

      Mount coverslips to slides.