Protocol for immunohistochemistry - paraffin sections

Deparaffinization and rehydration:

1. Incubate sections with xylene twice for 5 min.
   
   
2. Rehydrate sections through a series of ethanol concentration ranging from 100 % to 70 % (10 % steps) with a minimum of 5 min/concentration.
   
   
3. Rinse sections in PBS.
   

Antigen retrieval:

1. Microwave sections in 10 mM citrate buffer, pH 2.5, to retrieve antigens that are masked through the embedding process.
   
   
2. Cool slides slowly to RT.
   
   
3. Rinse slides in PBS.
   

Immunofluorescence:

1. Permeabilize sections on the slides with 0.5 % Triton X-100 (varies from antibody to antibody) in PBS for 10 min at RT.
   
   
2. Block in PBS / 2 % BSA / 0.5 % Triton X-100 for 1 h at RT.
   
   
3. Remove blocking solution and incubate sections with the primary antibody diluted in blocking solution overnight at 4°C.
   
   
4. Remove primary antibody and wash thoroughly with PBS / 0.5 % Triton X-100 3 times for 10 min.
   
   
5. Incubate with the secondary antibody for 1 h at RT.
   
   
6. Mount slices and examine microscopically.