IHC/ICC procedure for frozen sections, paraffin sections, cell smears.
1. Deparafinize and hydrate tissue sections through xylene or other clearing agents and graded alcohols.(For frozen sections or cell smears; use unfixed, acetone fixed or appropriate fixative for the antigen in question; for cell smears it may be necessary to permeabilize the cell by detergent, please refer to antibody protocol)
2. Wash 2-3 with distilled or deionized water.
3. Wash slide with Tris/saline buffer, followed by blocking with normal blocking serum, rinse with buffer.
4. Block with reagent A (Avidin) for 5-10 minutes, rinse with buffer.
5. Now block with reagent B (Biotin) for 5-10 minutes, wash thoroughly with buffer.
Note: If antigen retrieval is required it can be applied after this stage.
6. Wash slide with PBS or Tris saline (with 0.02-0.05% nonionic detergent, Triton X100, Tween 20 or NP-40)
7. Follow instructions for IHC/ICC.
1. After protein blocking step, soak blot in avidin reagent A, diluted 1:20 with tris/saline buffer for 5-10 minute.
2. Rinse blot with buffer; followed by soaking in diluted biotin reagent B (diluted 1:20) for 5-10 minutes.
3. Rinse with buffer.
These are guide lines, the optimum incubation times for these reagents and reactions should be determined by the individual lab.