Important: Some proteins have special requirements for good detection. Please refer to the remarks sections for IHC-P on the respective data sheet.
For the preparation of paraffin embedded tissues for immunohistochemistry, please refer to our tissue preparation protocols.
Materials and reagents
- Food steamer (e.g. Braun Multigourmet; alternatively: microwave, water bath, pressure cooker)*
- Staining containers with slide holders (e.g. Tissue-Tek)
- Blocking buffer: Protein Block Serum Free (Agilent cat. no. X0909)
- Antibody incubation buffer: Antibody diluent (Agilent cat. no. S2022)
- Biotinylated secondary antibody
- ABC HRP Kit: standard (Vectorlabs cat. no. PK-4000)
- ImmPACT DAB: (Vectorlabs cat. no. SK-4105)
- PBS: Phosphate buffered saline, (pH 7.4)
- TBST: Tris buffered saline with Tween 20, 20 mM Tris (pH 7.6), 150 mM NaCl, 0.05% Tween 20
- Antigen retrieval buffer: 10 mM citrate, 0.05% Tween 20, pH 6.0 or 10 mM Tris, 1 mM EDTA, 0.05% Tween 20, pH 9.0. Please check IHC-P remarks on the respective data sheet.
- Xylene, 100% ethanol, 90% ethanol, 80% ethanol and 70% ethanol, 2-propanol
- Optional: Hematoxylin Solution (Mayer's, Modified) or other nuclear counterstain
- Optional: Avidin/Biotin Blocking Kit (Vectorlabs cat. no. SP-2001)
- Non-aqueous mounting medium
Deparaffinization and rehydration
Deparaffinize and hydrate tissue sections
- Xylene 2x 5 min
- 100% EtOH 2x 2 min
- 90% EtOH 1x 2 min
- 80% EtOH 1x 2 min
- 70% EtOH 2x 2 min
- Deionized Water 1x 20 sec
- PBS 1x 2 min
Keep the slides in PBS until ready to perform the Antigen Retrieval. Do not allow the slides to dry out.
Antigen retrieval (using a food steamer)*
- Heat the steamer with a suitable staining container filled with Antigen retrieval buffer to ~97°C.
- Transfer the sections into the staining box, wait until the temperature reaches 97°C.
- Incubate the sections in the steamer for 30 min.
- Remove the staining container from the steamer and allow the slides to cool down for 20 min (target end temperature ~60°C).
- Wash slides in PBS, 3x 1 min.
- Incubate the sections with 3% hydrogen peroxide in PBS (freshly prepared!) for 5 min to block endogenous peroxidase activity.
- Wash slides in PBS, 2x 1 min.
- Wash slides in TBST, 1x 2 min.
- Optional: Some antibodies require an additional antigen retrieval step with formic acid. Please check IHC-P remarks on the respective data- or factsheet. If formic acid treatment is required, incubate slides for 3 min in 88% formic acid. Wash slides in TBST, 3x 1 min.
- Optional: Perform Avidin-Biotin-Block according to manufacturer’s instructions.
Note: Certain tissues (e.g. liver, kidney) contain high levels of endogenous biotin. The Avidin-Biotin blocking step is recommended when using the ABC system for these tissues. If the background problem persists, consider trying a polymer-based detection system instead of biotinylated secondary antibody/ABC system.
- Block in blocking buffer for 10 min.
- Drain slides (do not rinse).
- Apply primary antibody diluted in antibody incubation buffer and incubate in a humidified chamber for 1 h at room temperature.
- Wash slides in TBST, 3x 2 min.
- Apply secondary antibody diluted in antibody incubation buffer for 30 min at room temperature.
- In the meantime, prepare the ABC-reagent: 5 ml PBS + 1 drop A + 1 drop B and incubate for 30 min.
- Apply the ABC reagent for 30 min at room temperature.
- Wash slides in TBST, 3x 2 min.
Chromogenic detection with DAB
- Apply the DAB substrate for 1-10 min.
Note: Observe the staining with a microscope! Development times may differ depending upon the level of antigen.
- Stop the DAB reaction with deionized water.
- Follow the manufacturer’s instructions for counterstaining and bluing.
- Wash slides in deionized water for 1 min.
Dehydration and mounting
- 70% EtOH 2x 10 sec
- 80% EtOH 1x 10 sec
- 90% EtOH 1x 10 sec
- 2-Propanol 3x 1 min
- Xylene 3x 2 min
Mount slides in a suitable organic mounting medium and add coverslip.
*For an alternative Antigen Retrieval protocol using a water bath check protocol-ihc-paraffin-fluorescent.
Note: The SYSY standard protocol generates good staining results in the SYSY labs and may be used as suggestion. However, to achieve the highest specific signal and lowest non-specific background signal, the best antigen retrieval condition, antibody concentration, incubation temperature and incubation time must be determined individually.