This article contains IHC protocol for Paffain sections. immunohistochemistry protocol (paraffin sections).

Fixation and Sectioning

1. Fix dissected tissues in 2% paraformaldehyde, Bouin’s solution, or other fixative for 30 minutes to overnight. (NOTE: Fixation time will depend on species, tissue origin and size of tissue sections).
2. Embed tissue in paraffin.
3. Section the tissues 5-10 micrometers thick.
4. If using paraformaldehyde as fixative antigen retrieval may be necessary.

Deparaffinization and Tissue Rehydration

1. Deparaffin samples by incubating sections 2-3 times in xylene for 10 minutes each.
2. Hydrate samples by placing in 100% ethanol for 2 x 3 minutes each, then in 95%, 70%, 50%, 30% ethanol for 1 x 2 minutes each.

Blocking and Primary Antibody Incubation

1. Block slides with 10% serum from the species from which the secondary antibody was taken or 8% BSA.
2. Incubate for 30 minutes to 1 hour at room temperature in a humidified chamber.
3. Wash samples in PBS containing 2% BSA for 5 minutes.
4. Incubate slides in a humidified chamber overnight with primary antibody solution (primary antibody in PBS containing 2% BSA).
5. Wash three times with PBS containing 2% BSA for 5 minutes.

Secondary Antibody Incubation and Detection

1. Incubate slides in a humidified chamber with secondary antibody solution (secondary antibody in PBS containing 2% BSA) according to manufacturer’s protocols.
2. Wash three times with PBS containing 2% BSA for 5 minutes.
3. Detect according to manufacturer’s protocols.

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