Protocol/Immunoradiography

Protocol for Immunoradiography

Materials and Reagents

• Appropriate primary polyclonal antibody

raised in rabbit against gene of interest.

• Cryostat (Leica).
• Cryostat sections (15–20 μm thick)

cut at -20°C.

• Cromallun–gelatin-coated slides (see

above).

• Dessicator.
• PFA (see above).
• PBS.
• BSA.
• Normal goat serum (Sigma).
• Donkey antirabbit [35S]IgG (200–

2000 Ci/mmol, 100 μCi/mL; Amersham Pharmacia Biotech).

• β max films (Amersham Pharmacia

Biotech).

• Computerized image analysis system

(Biocom, Les Ulis, France).

Procedure

This protocol is adapted from Reference 4. 1. The cryostat sections are dessicated at 4°C and frozen at -80°C until used. After a 3-minute fixation (4% paraformaldehyde in PBS) at 4°C sections are preincubated for 1 hour in PBS supplemented with 3% BSA and 1% goat serum, then incubated overnight in an appropriate concentration of polyclonal primary antibody raised in rabbit. 2. After extensive washes in PBS, sections are incubated for 2 hours at room temperature in donkey antirabbit [35S]IgG. 3. After abundant washing, sections are air-dried and apposed to β max film for 1 to 2 days. 4. Optical density is measured by computerized image analysis.

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