IHC/ICC procedure for frozen sections, paraffin sections, cell smears and immunoblots.
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 S (streptavidin) 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.
WB Procedure
1. After protein blocking step, soak blot in streptavidin reagent S, 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.
