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2-Step Immunofluorescence Protocol: Cells Grown in Culture and Cytospin Preparations

See the 2-step immunofluorescence protocol to be used for cells grown in culture and cytospin preparations. Includes required reagents, preparation steps and procedure.

Materials

  • Acetone
  • Hydrophobic barrier pen
  • Wash Solution
  • Normal Goat Serum Blocking Solution
  • IHC Antibody Diluent
  • Goat anti-Rabbit IgG Antibody-DyLight conjugated (Bethyl cat# A120-101D2, A120-101D3, A120-101D4, or cross-adsorbed antibodies A120-201D2, A120-201D3, A120-201D4)
  • Fluorescent mounting media with or without DAPI
  • Coverglass

Preparation of Materials


Normal Goat Serum Blocking Solution

  • 20 ml normal goat serum (Filter sterilize with 0.2 µM filter)
  • Volume to 100 ml with PBS or TBS

Store 2-8 °C, discard after 3 months.


IHC Antibody Diluent (50mM TBS, 1% BSA)

  • 200ml 1X TBS
  • 2 g BSA (Sigma A9647)
  • Volume to 100 ml with PBS or TBS.

Store 2-8 °C, discard after 1 year.

*May want to use a perservative.


Wash solution

Can be any of the following: PBS, TBS or 0.01% Tween 20 in dH20. TBS wash solution is recommended for phospho antibodies.


Secondary Antibody

Goat anti-Rabbit IgG Antibody-Dylight conjugated. (Bethyl cat# A120-101D2, A120-101D3, A120-101D4, or cross-adsorbed antibodies A120-201D2, A120-201D3, A120-201D4) Dilute 1:100 with IHC antibody diluent. Prepare just prior to use. Protect from light. Optimal working dilutions should be determined experimentally by the investigator.


Procedure

  1. For cells in chambered microscope slides or cells grown on coverslips: Gently rinse off media with PBS 3 changes for 1 minute each. For cytospins: Allow to air dry after preparation.
  2. Actone fixation (ice cold) 10 minutes
  3. Air dry 30 minutes in hood
  4. Circle cytospin with a hydrophobic barrier pen.
  5. Wash solution. Do not allow sections to dry for the remaining procedure.
  6. Normal Goat Serum Blocking Solution - 15 minutes
  7. Primary Antibody Incubation: 30 minutes room temperature.
    • Prepare primary antibody with IHC Antibody Diluent.
    • Optimal working dilutions should be determined experimentally by the investigator.
    • Refer to Bethyl antibody datasheet for suggested dilution range.
  8. Wash solution- 3 changes for 5 minutes each
  9. Secondary Antibody Incubation: 30 minutes - room temperature. Protect from light.
  10. Wash Solution - 3 changes for 5 minutes each
  11. Remove chamber from microscope slide
  12. Mount with fluorescent mounting media and coverslip. Use fluorescent mounting media with DAPI if counterstaining is desired.