IHC/ICC procedure for frozen sections, paraffin sections, cell smears and immunoblots.
- 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 permealize the cell by detergent, please refer to antibody protocol)
- Wash 2-3 with distilled or deionized water.
- Wash slide with Tris/saline buffer, followed by blocking with normal blocking serum, rinse with buffer.
- Block with reagent S (streptavidin) for 5-10 minute, rinse with buffer.
- Now block with reagent B (Biotin) for 5-10 minutes, wash thoroughly with buffer.
Note: If antigen retriever is required it can be applied after this stage.
- Wash slide with PBS or Tris saline (with 0.02-0.05% nonionic detergent, Triton X100, Tween 20 or NP-40) or washing buffer (Immuno Automation buffer IBSC cat # AR-6561) 3-5X. Wash slide with PBS 1X.
- Follow instructions for IHC/ICC.