Immunohistochemistry (IHC)

IHC stainings with chromogenic or fluorescent detection

1. Cut skin or tumor biopsy longitudinally with scalpel

2. Fix tissue for 24 h at 4°C in 4% buffered formalin. For skin or tumors surrounded by normal skin, pin the skin to cork with slight tension prior to fixing, as skin tends to curl

3. Embed fixed tissue in paraffin

4. Using a microtome, prepare 3 µm thick sections

5. Deparaffinize sections

            5.1 100% xylol for 5 min

            5.2 100% xylol for 5 min

            5.3 100% ethanol for 10 min

            5.4 90% ethanol for 5 min

            5.5 70% ethanol for 5 min

            5.6 PBS for 5-10 min

6. Perform HIER in citrate buffer pH6.0 (10 mM citrate)

            7.1 Boil 10 min in a steam pot; cool down in steam pot for 15 min

            7.2 Transfer to PBS for 1 min

7. Circle the section with Marabu Fixogum or Liquid Blocker Pen

8*. Block endogenous peroxidases with Dako REAL peroxidase blocking solution (Agilent Technologies) for 5 min at RT and rinse slides in PBS for 1 min with a magnetic stirrer

9*. Blocking of endogenous avidin and biotin with Avidin/Biotin Blocking Kit (Vectorlabs)

            9.1 Apply avidin block for 10 min at RT

            9.2 Rinse the slides in PBS for 1 min with a magnetic stirrer

 9.3 Apply biotin block for 10 min at RT

            9.4 Rinse the slides in PBS for 1 min with a magnetic stirrer

10. Block sections for 1 h at RT with 5% goat serum/5% FCS/1% BSA in PBS

11. Incubate overnight at 4°C in a wet chamber with primary antibody diluted in 5% goat serum/5% FCS/1% BSA in PBS

*This step is not necessary for fluorescence stainings

Chromogenic detection via Dako REAL™ Detection System, Peroxidase/AEC, Rabbit/Mouse

12. Incubate sections 20 min at RT with the Multilink secondary Antibody (goat anti-rabbit, goat anti-mouse) from Dako REAL Kit bottle A

13. Rinse slides for 1 min at RT in PBS-T (0.5% Tween-20) with magnetic stirrer

14. Incubate sections 20 min at RT with the HRP from Dako REAL Kit bottle B

13. Rinse the slides for 1 min at RT in PBS-T (0.5% Tween-20) with magnetic stirrer

14. Detect proteins under the microscope by application of AEC from Dako REAL Kit bottle C until a convenient staining is achieved (2-15 min depending on the antibody and the target)

15. Stop color reaction by submerging the section in ddH₂O

16. Counterstain with Mayer′s hemalum solution (some seconds, but depending on the strength of the target staining and the company used) and stop staining by submerging in flowing TAP water

17. Mount sections with Dako Faramount Aqueous Mounting Medium

Detection via fluorescence

12. Wash sections 3 times with PBS-T (0.5% Tween-20)

13. Incubate sections for 45 min at RT with species-specific Alexa Fluor-conjugated secondary antibody diluted in 1% goat serum/PBS (Invitrogen, 1:1000)

14. Wash sections 2 times with PBS

15. Stain nuclei for 5 min at RT with DAPI (0.3 μg/ml in PBS) prior to 4 washes for 2 min in PBS

16. Rinse shortly with ddH₂O to remove salts from PBS

17. Mount sections with Dako Faramount Aqueous Mounting Medium

Primary antibodies:

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