IHC On Paraffin Sections

Step-by-step protocol and tips for immunohistochemistry on paraffin-embedded sections.

Immunohistochemistry On Paraffin Sections

Please note: All steps in the following protocol are carried out at room temperature unless stated otherwise.

Deparaffinizing and rehydration

1. Immerse slides in xylene for 10 minutes. Repeat this step again in fresh xylene for a further 10 minutes.

   (If required, repeat a third time in fresh xylene for another 10 minutes.)

2. Rehydrate sections by sequentially incubating with 100%, 95%, 80%, and 60% ethanol for 5 minutes each.

3. Rinse sections with distilled water three times for 3 minutes each.

Heat-induced Epitope Retrieval (HIER) (optional) 

1. Transfer sections into a container and cover with enough citrate buffer pH 6.0 *

2. Heat in a microwave at the medium power for 10 min.

3. Allow slides to cool in the buffer for approx 35 min.

   * or Tris-EDTA buffer (pH9). Optimal conditions always have to be determined by each laboratory and by the specific product information.

Primary antibody incubation

1. Rinse slides three times with 1x TBS for 3 minutes each.

2. Incubate slides with 3% H₂O₂ solution (diluted in distilled water) for 10 minutes to quench endogenous peroxidase activity.

3. Rinse slides three times with 1x TBS for 3 minutes each.

4. Prepare 5% normal blocking serum in 1x TBS. The serum should be derived from the same species in which the secondary antibody was raised. Block the sections for 1 hour.

(Alternatively, use 5% BSA in 1x TBS for blocking if the corresponding serum is not available.)

1. Incubate sections with the primary antibody diluted in 1x TBS for 1.5 hours, or overnight at 4ºC; the optimal antibody dilution ratio should be pre-determined by experimentation. Set up negative controls by omitting the primary antibody incubation step for one slide per each experimental condition.

2. Following primary antibody incubation, rinse slides three times with 1x TBS for 3 minutes each.

Signal Detection

Please note: Proteintech®* routinely uses EnVision Kit (Dako) for this step.

1. Apply sufficient peroxidase labeled polymer and incubate for 30 minutes.

2. Rinse slides three times with 1x TBS for 3 minutes each.

3. Prepare an appropriate volume of substrate solution prior to use by mixing one drop of Liquid DAB plus chromogen immediately with 1 ml of substrate buffer. Apply the substrate carefully and incubate for 5–10 minutes until a brown colour develops.

4. Rinse sections gently with sufficient distilled water.

Hematoxylin counterstaining (optional)

1. To stain nuclei, immerse slides in a bath of hematoxylin for 3 minutes.

2. Rinse slides gently with distilled water.

3. Transfer slides into a 1% HCI, 99% ethanol solution for 10 seconds; transfer to distilled water immediately.

Dehydration and mounting

1. Immerse slides sequentially into 60%, 80%, 95%, and 100% ethanol baths for 5 minutes each.

2. Immerse slides in xylene for 5 minutes. Repeat this step again in fresh xylene for another 5 minutes.

3. Mount the section with sufficient mounting media and cover with a coverslip Air-dry in a well-ventilated area (e.g., fume hood).

Tip: Metal ions (e.g. CuSO4, methenamine silver, cobalt chloride, ammonium nickel sulfate, nickel chloride) can enhance IHC signal.

Tip: When DAB staining is nuclear, shorten the counterstaining incubation time and wash the slide with ammonia or TBS buffer (pH 8.0).