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2-Step Immunofluorescence Protocol: Formaldehyde-Fixed Cells and Cytospin Preparations

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

For antibodies made in Rabbit

Materials:

  • 3-4% Paraformaldehyde (freshly prepared) or 10% Neutral Buffered Formalin
  • Triton-X 100
  • 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 Reagents:

Norml 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)

190 ml 1X TBS
2 g BSA (Sigma A9647)
Volume to 200 ml with 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.


10% Triton-X 100 Stock

10 ml Triton-X 100
90 ml dH20


0.25% Triton-X 100

25 µl 10% Triton-X 100
1 ml TBS


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 TBS - 3 changes for 1 minute each. For cytospins: Allow to air dry for 1-2 minutes after preparation.
  2. Formaldehyde fixation - 10-30 minutes
  3. Wash Solution - 3 changes for 5 minutes each
  4. Circle cytospin with a hydrophobic barrier pen. Do not allow cells to dry for the remaining procedure.
  5. Permeabilization step: 0.25% Triton-X 100 - 2-10 minutes. Optimal working dilution and incubation time should be determined experimentally by the investigator.
  6. Wash Solution - 3 changes for 5 minutes each
  7. Normal goat serum Blocking Solution - 15 minutes
  8. Primary Antibody Incubation: 30 minutes - room temperature. Prepare primary antibody with IHC Antibody Diluent. Optimal working dilutions should be determined experimentally by the investigator.
  9. Wash Solution - 3 changes for 5 minutes each
  10. Secondary Antibody Incubation: 30 minutes - room temperature. Protect from light.
  11. Wash Solution - 3 changes for 5 minutes each
  12. Remove chamber from microscope slide.
  13. Mount with fluorescent mounting media and coverslip. Use fluorescent mounting media with DAPI if counterstaining is desired.

NOTE: This procedure is suitable for Phospho-specific Antibodies

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