This Immunohistochemistry protocol is for frozen sample sections.

Fixation and Sectioning

1. Freeze and section tissue samples.
2. Fix sections in Bouin’s solution, -20°C acetone, -20°C methanol, or formaldehyde for 2-30 minutes. (NOTE: Fixation time will depend on species, tissue origin and size of tissue sections).

Blocking and Primary Antibody Incubation

1. Block slides with 10% serum from the species of the secondary antibody 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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