Immunofluorescence on adherent cells using anti-phosphatidylinositol (PI) antibodies

Immunofluorescence on adherent cells using anti-phosphatidylinositol (PI) antibodies.

Abstract

In this protocol, adherent HeLa cells are fixed to glass coverslips and stained by specific antibodies targeting phosphoinositides (i.e. Purified Anti-PtdIns(3,4)P2 IgG, Z-P034, Echelon) and fluorescently conjugated secondary antibodies.

Keywords: Immunofluorescence, Phosphoinositide, Cell, Adherent

Materials and Reagents

1. Hela cells
2. Phosphate Buffer Saline (PBS).
3. Bovine Serum Albumin (BSA).
4. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 158127).
5. Triton X-100 (Sigma-Aldrich, catalog number: T8787).
6. Saponin (Sigma-Aldrich, catalog number: SAE0073).
7. Primary antibody (i.e. Purified Anti-PtdIns(3,4)P2 IgG, Z-P034, Echelon).
8. Secondary antibody (Alexa Fluor^® Antibodies).
9. Anti-fade mounting medium: i.e. ProLong Gold Antifade Mountant (Thermo Fisher, catalog number: P10144) with DAPI (if nuclear staining is needed) or without DAPI.

Equipment

1. Round coverslips (Thermo Fischer).
2. 24-well plate.
3. PARAFILM^® M (Sigma-Aldrich, catalog number: P7793).
4. Fluorescence microscope.

Procedure

1. Grow cells on round coverslips in 24-well plates to 40-60% confluence. Do not overgrow because it will be difficult to distinguish cell components when stained.
2. Wash cells with 500 μl/well PBS twice, then add 350 μl/well 2% PFA at room temperature (RT) for 15 min.
3. Remove PFA and wash the cells with 500 μl/well PBS for 3 times.
4. Permeabilize the cells with either: 350 μl/well PBS + 0.1% Triton X-100 for 5 min at RT or 350 μl/well PBS + 0.5% saponin for 20 min at RT.
5. Remove permeabilization solution and wash the cells with 500 μl/well PBS for 3 times.
6. Add 350 μl/well of blocking solution (1% BSA in PBS) for 30 min at RT.
7. Remove blocking solution and transfer the glass coverslips on PARAFILM^® M (Sigma-Aldrich) in a humidified chamber.
8. Add 60 μl/coverslips of blocking solution containing 5 μg/mL of primary antibody (i.e. Anti-PI(3,4)P2 Antibody, Z-P034, Echelon) and incubate for 2 hours at room temperature in a humidified chamber.
9. Remove primary antibody-containing solution and wash 3 times with 60 μl/coverslips of PBS + 1% BSA for 5 min.
10. Add 60 μl/coverslips of blocking solution containing 2 μg/mL of secondary antibody (i.e. Alexa Fluor® 568, Thermo Fisher) and incubate in the dark for 1 hour at room temperature in a humidified chamber.
11. Remove secondary antibody-containing solution and wash 3 times with 60 μl/coverslips of PBS for 5 min.
12. Place a small drop of anti-fade reagent (i.e. ProLong Gold Antifade Mountant-Life Technologies, Invitrogen^TM, with or without DAPI to stain nuclei) on a glass slide, then get the coverslip from the humidified chamber and put it face down on the drop, push it tightly (i.e. with the back of pipette tip) and attach to the glass slide.
13. Leave the slide in the dark for at least 30 min to let it dry at RT.
14. The slide now is ready to observe under a fluorescence microscope or can be stored in the dark at 4 C for no longer than 2 days.

Alternative Procedures and Precautions

• For chemical fixation of the cells, PFA is preferable over methanol as it does not dehydrate cells or tissue. If needed, i.e. for co-staining with primary antibodies targeting microtubules and the tubulin cytoskeleton, methanol fixation can be performed for 2-3 min at -20 C. It is always recommendable to compare the staining results with a crosslinker and solvent for the phosphoinositide of interest.

• To quench free aldehyde groups from fixation before cell permeabilization, cells can be washed 3 times in PBS with 50mM NH4C 50mM ammonium chloride (50 mM NH₄Cl).

• For permeabilization: Triton X-100 is a non-ionic detergent, non-specific that will generally permeabilize all cellular membranes. It may damage structure likes Golgi apparatus or the endoplasmic reticulum. However, it is the only common permeabilization reagent that can effectively penetrate the nuclear membrane. Saponin is amphipathic and selective for cholesterol. This selectivity allows for efficient permeabilization of the plasma membrane but not of some other organelles like the nucleus or mitochondria.

• For blocking solution and primary/secondary antibody incubation: because saponin’s effects may be reversible and possibly lost to washes, its use can be maintained throughout the staining protocol at a concentration comprised between 0,5-0,1%, to ensure better antibody access.

• For nuclear staining: TO-PRO-3 Stain (Thermo Fisher, T3605) can be preferred to DAPI because of its increased sensitivity and photostability.
  1. After washing the secondary antibody, add 60 μl/coverslips of solution containing TO-PRO-3 diluted 1:2000 in PBS. Incubate in the dark in a humidified chamber for 15 min.
  2. Remove TO-PRO-3-containing solution and wash 3 times with 60 μl/coverslips PBS for 1 min.
  3. Proceed with protocol as at point 12.

• Anti-phosphoinositide antibodies (i.e. Purified Anti-PtdIns(3,4)P2 IgG, Z-P034, Echelon) can be stored at 4 C for a few weeks. Alternatively, aliquot and store at -20 avoiding freeze/thaw cycle that may irreversibly damage the antibody.

Acknowledgments

This protocol was adapted from (1) and developed at the Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy.

For further information and additional methods applicable to other anti-phosphoinositide antibodies and for staining of specific subcellular compartments, please refer to the following publications (2-6) and to the Echelon webpage (https://echelon-inc.com/wp-content/uploads/2020/07/TDS_Z-ICCL_Rev2.pdf).

References

1.           Y. Posor et al., Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate. Nature499, 233-237 (2013).

2.           G. R. Hammond et al., PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity. Science337, 727-730 (2012).

3.           Gerald R. V. Hammond, G. Schiavo, Robin F. Irvine, Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P2. Biochemical Journal 422, 23-35 (2009).

4.           A. L. Marat et al., mTORC1 activity repression by late endosomal phosphatidylinositol 3,4-bisphosphate. Science356, 968-972 (2017).

5.           W. Elong Edimo et al., SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells. J Cell Sci 129, 1101-1114 (2016).

6.           G. R. Hammond et al., Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells. J Cell Sci 119, 2084-2094 (2006).

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