General Immunofluorescent Protocol for Millicell® Products

Immunocytochemical staining is a technique employing fluorescently labeled antibody, by which cells can be localized, labeled, and examined via fluorescent microscopy.

Materials and Reagents

  • Millicell® Cell Culture Inserts and Insert Plates
  • Sterile Phosphate Buffer Saline (PBS)
  • Methanol, 100%
  • Glycerol
  • FITC-conjugated antibody
  • 1% BSA in PBS
  • Glass slides

Method

  1. Aspirate cell culture media and wash Millicell® inserts or plates gently on both sides with PBS. Incubate for 5 minutes and repeat 2–3X. Consult the recommended working volumes table for appropriate volumes.

  2. Add fixative solution (e.g. methanol) for 5–10 minutes. It is not required to treat the underside of the membrane with fixative. Incubate according to protocol instructions.

  3. After treatment, aspirate fixative and fill filter wells with washing buffer. Repeat 2–3X in order to fully remove the fixative solution from both sides of the filter membrane. Do not allow cells to dry.

  4. Dilute primary antibody according to vendor recommendations. In order to obtain best results, it is recommended that optimal working dilutions be determined by the user. If permeabilization is required (such as for cytoplasmic or nuclear antigens), saponin can be added to the solution at a concentration of 0.1%

  5. Add antibody solution to each well then incubate at recommended temperature (typically room temperature or 4°C) with mild shaking or rocking to assure that solution wets out entire filter surface. If antibody is fluorescently labeled (direct labeling), cover plate with foil to protect from light.

  6. Aspirate antibody solution and wash both sides of membrane as indicated in Step 1 to remove all unbound antibody.

  7. If performing indirect labeling with a secondary antibody, repeat steps 4 through 6 with the secondary antibody. For visualization using fluorescent antibodies, continue to Visualization and Microscopy procedures. For enzyme linked assays (HRP, etc.), follow vendor procedures for developing.

Materials