IHC On Paraffin Sections
Immunohistochemistry On Paraffin Sections
Deparaffinizing and rehydration
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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.) -
Rehydrate sections by sequentially incubating with 100%, 95%, 80%, and 60% ethanol for 5 minutes each.
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Rinse sections with distilled water three times for 3 minutes each.
Heat-induced Epitope Retrieval (HIER) (optional)
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Transfer sections into a container and cover with enough citrate buffer pH 6.0 *
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Heat in a microwave at the medium power for 10 min.
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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
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Rinse slides three times with 1x TBS for 3 minutes each.
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Incubate slides with 3% H₂O₂ solution (diluted in distilled water) for 10 minutes to quench endogenous peroxidase activity.
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Rinse slides three times with 1x TBS for 3 minutes each.
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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.)
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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.
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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.
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Apply sufficient peroxidase labeled polymer and incubate for 30 minutes.
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Rinse slides three times with 1x TBS for 3 minutes each.
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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.
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Rinse sections gently with sufficient distilled water.
Hematoxylin counterstaining (optional)
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To stain nuclei, immerse slides in a bath of hematoxylin for 3 minutes.
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Rinse slides gently with distilled water.
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Transfer slides into a 1% HCI, 99% ethanol solution for 10 seconds; transfer to distilled water immediately.
Dehydration and mounting
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Immerse slides sequentially into 60%, 80%, 95%, and 100% ethanol baths for 5 minutes each.
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Immerse slides in xylene for 5 minutes. Repeat this step again in fresh xylene for another 5 minutes.
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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). |
