Binding Antibodies to Attached Cells or Tissues in Preparation for Staining

Abstract

Cells for staining are usually prepared from one of three sources: adherent cells, suspension cells, or whole tissues. Antibodies generally are applied directly to the area of the cells or tissues that is being studied. The antibodies can be labeled directly or they can be detected by using a labeled secondary reagent that will bind specifically to the primary antibody. Detection reagents for cell staining can be labeled with fluorochromes, enzymes, gold, or iodine. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. ReagentsCells or tissues that have been attached to a substrate (coverslips, slides, or plates), fixed, and washed Phosphate-buffered saline, supplemented with 1% Triton X-100 or NP-40 if necessary See Step 4. Primary antibody solution, diluted (see Step 2)All dilutions of the primary antibody must be performed in protein-containing solutions. For example, use phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA).

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“…Xenografts fixation and immunohistochemistry were performed as described previously [ 17 , 18 ]. Briefly, 4 µm paraffin sections were dewaxed with xylene, rehydrated with graded alcohol series and heated with citrate buffer.…”