Developmental Biology Immunology Cell Culture

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Authors: Mohamed Kodiha, Rehan Umar & Ursula Stochaj

Introduction

Indirect immunofluorescence is a sensitive method to monitor the distribution of proteins. However, frequently a protein appears to be in distinct locations, even when analyzed in the same cell type under identical conditions. One possible cause that contributes to these discrepancies is the protocol used for immunodetection (1-3). Despite these complications, differences in staining can be useful to gain insight into protein functions associated with a specific subcellular location. The nucleoporin Nup98 is a compelling example for which the methodical comparison of staining protocols provides the basis to monitor its interaction with distinct compartments. We have chosen Nup98 as it is a mobile protein which resides in the cytoplasm, the nuclear interior, as well as the nuclear and cytoplasmic sides of NPCs (4-6). Within the nucleus, Nup98 associates not only with GLFG bodies in the nucleoplasm but also with nucleoli. Nup98 is a key component in nuclear transport (7-10) and an important factor in cancer research (11). As mislocalization of Nup98 fusion proteins correlates frequently with human leukemia, reliable methods are required that detect Nup98 in different locations. The protocol described here can be used to evaluate systematically and optimize the detection of proteins that are present in multiple cellular compartments.

Reagents

  1. Acetone
  2. Bovine serum albumin (BSA)
  3. Cell culture medium (Gibco)
  4. Formaldehyde
  5. Methanol
  6. NaN3
  7. PBS, phosphate-buffered saline, pH 7.4
  8. Triton X-100
  9. Tween 20
  10. Antibodies against the protein of interest; here: polyclonal anti-Nup98 (Santa Cruz Biotechnology, sc-14153)
  11. Cy3-labeled affinity-purified secondary antibodies (Jackson ImmunoResearch).
  12. 4’,6-diamidino-2-phenylindole (DAPI; Sigma)
  13. Vectashield (Vector Laboratories)

Equipment

  1. CO2 incubator
  2. Laminar flow hood
  3. Humidity chamber; assembled from pipet tip box and styrofoam supports
  4. Confocal microscope (for example: Zeiss LSM510) or fluorescence microscope equipped with appropriate camera

Procedure

CELL CULTURE

Culturing of HeLa cells on poly-lysine coated coverslips has been described previously (12,13).

INDIRECT IMMUNOFLUORESCENCE

All steps are carried out in a humidity chamber.

Option 1

Methanol/acetone fixation.

  1. Fix in methanol/acetone 1:1 (vol/vol) for 30 min at -20 °C. All subsequent steps are at room temperature.
  2. Dry for 15 min and rehydrate with PBS for 15 min.
  3. Block non-specific binding sites with PBS/2 mg/ml BSA for 1 h.
  4. Incubate overnight with anti-Nup98 (0.5 µg/ml) in blocking solution.
  5. Next day, wash 3 times for 10 min with PBS/2 mg/ml BSA. Incubate 2 h with secondary antibodies (2 μg/ml) in blocking solution.
  6. Wash 3 times for 10 min in blocking solution; incubate with 1 μg/ml DAPI in blocking solution for 2 min.
  7. Mount specimen in Vectashield and seal with nail polish.

Option 2

Formaldehyde fixation.

  1. All steps are carried out at room temperature. Wash once with PBS and fix with 3.7% formaldehyde in PBS for 25 min.
  2. Rinse with PBS, and then permeabilize with 0.1% Triton X-100 in PBS/2 mg/ml BSA/1 mM NaN3 for 5 min.
  3. Block with 0.05% Tween 20 in PBS/2 mg/ml BSA/1 mM NaN3 (blocking solution) for 1 h.
  4. Incubate overnight with anti-Nup98 (0.5 µg/ml) in blocking solution.
  5. Wash 3 times for 10 min with blocking solution, then incubate for 2 h with secondary antibodies (2 μg/ml) in blocking solution.
  6. Wash 3 times for 10 min with blocking solution, then incubate with 1 μg/ml DAPI in blocking solution for 2 min.
  7. Mount specimen in Vectashield and seal with nail polish.

Option 3

Formaldehyde fixation.

  1. Wash once with PBS and fix with 4% formaldehyde in PBS for 10 min at room temperature.
  2. Rinse with PBS, then permeabilize with PBS/ 0.2% Triton X-100/2 mg/ml BSA/1 mM NaN3 on ice for 10 min. All subsequent steps are carried out at room temperature.
  3. Block with PBS/0.02% Triton X-100/3% BSA/1 mM NaN3 (blocking solution) for 30 min.
  4. Incubate with anti-Nup98 (2 µg/ml) in blocking solution for 30 min.
  5. Wash 4 times with PBS/0.02% Triton X-100/1.5% BSA/ 1 mM NaN3; then incubate with secondary antibodies (20 μg/ml) in blocking solution for 30 min.
  6. Wash 4 times with PBS/0.02% Triton X-100/1.5% BSA/ 1 mM NaN3. Incubate with 1 μg/ml DAPI in washing solution for 2 min.
  7. Mount specimen in Vectashield and seal with nail polish.

Option 4

Formaldehyde fixation.

Same as protocol 3 except for the antibody concentrations and incubation periods (0.5 µg/ml anti-Nup98, overnight incubation) and (secondary antibodies 2 µg/ml for 2 h).

IMAGE ACQUISITION

Acquire images by confocal microscopy (for example, Zeiss LSM510) using a 63x objective (oil, NA = 1.4), pixel resolution of 0.65 µm, scan speed 5, four-line averaging (13).

PROCESSING OF IMAGES

Process images with appropriate software such as Adobe Photoshop 8.0.

Timing

  • Staining option 1: 20 hours
  • Staining option 2: 20 hours
  • Staining option 3: 3.5 hours
  • Staining option 4: 20 hours

Critical Steps

Optimization of antigen detection

The optimal concentrations of primary and secondary antibodies have to be determined for each individual protein.

Incubation of coverslips in humidity chamber

To obtain low background and even staining it is essential to keep samples moist throughout the procedure. We use empty pipet boxes and fill the bottom chamber with distilled water. Coverslips size 18×18 mm are placed on styrofoam supports (10×10 mm) that are glued to the grid. Keeping coverslips on these supports prevents the loss of solutions throughout the incubation period.

Troubleshooting

Non-specific staining or high background

  1. Use affinity-purified antibodies.
  2. Always determine the signal obtained for secondary antibodies only under identical staining conditions.
  3. Remove aggregates by centrifugation (5 min at 13,000 rpm, microfuge) of diluted primary and secondary antibodies immediately before use.

No staining

Many of the commercially available antibodies have not been tested for immunofluorescence, although the manufacturer recommends them for this procedure. It is useful to determine whether the supplier has characterized the antibody and can provide images for staining.

Anticipated Results

Option 1: Preferential detection of Nup98 associated with nucleoli.

Option 2: Staining of Nup98 associated with nuclear bodies and nucleoli, some staining of cytoplasmic Nup98.

Option 3: Staining of Nup98 associated with nuclear bodies and nucleoli, some staining of cytoplasmic Nup98.

Option 4: Preferential detection of cytoplasmic Nup98.

References

  1. Vekemans, K., Rosseel, L., Wisse, E. & Braet, F. Immuno-localization of Fas and FasL in rat hepatic endothelial cells: influence of different fixation protocols. Micron 35, 303-306 (2004).
  2. Morrissey, A., Rosner, E., Lanning, J., Parachuru, L., Dhar Chowdhury, P., Han, S., Lopez, G., Tong, X., Yoshida, H., Nakamura, T.Y., Artman, M., Giblin, J.P., Tinker, A. & Coetzee, W.A. Immunolocalization of KATP channel subunits in mouse and rat cardiac myocytes and the coronary vasculature. BMC Physiol. 12, 5 (2005).
  3. Bos P.K., van Osch, G.J.V.M., van der Kwast, T., Verwoerd-Verhoef, H.L. & Verhaar J.A.N. Fixation-dependent Immunolocalization Shift and Immunoreactivity of Intracellular Growth Factors in Cartilage. J. Mol. Histol. 32, 391-396 (2000).
  4. Griffis, E.R., Craige, B., Dimaano, C., Ullman, K.S. & Powers, M.A. Distinct functional domains within nucleoporins Nup153 and Nup98 mediate transcription-dependent mobility. Mol. Biol. Cell. 15, 1991-2002 (2004).
  5. Griffis, E.R., Altan, N., Lippincott-Schwartz, J. & Powers, M.A. Nup98 is a mobile nucleoporin with transcription dependent dynamics. Mol. Biol. Cell. 13, 1282-1297 (2002).
  6. Griffis, E.R., Xu, S. & M.A. Powers. Nup98 localizes to both nuclear and cytoplasmic sides of the nuclear pore and binds to two distinct nucleoporin subcomplexes. Mol. Biol. Cell. 14, 600-610 (2003).
  7. Powers, M.A., Macaulay, C., Masiarz, F. & Forbes, D.J. Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication. J. Cell Biol. 128, 721-736 (1995).
  8. Radu, A., Moore, M.S. & Blobel, G. The peptide repeat domain of nucleoporin Nup98 functions as docking site in transport across the nuclear pore complex. Cell 81, 215-222 (1995).
  9. Powers, M.A., Forbes, D.J., Dahlberg, J.E. & Lund, E. The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways. J. Cell Biol. 136, 241-250 (1997).
  10. Blevins, M.B., Smith, A.M., Phillips, E.M. & Powers, M.A. Complex formation among the RNA export proteins Nup98, Rae/Gle2, and TAP. J. Biol. Chem. 278, 20979-20988 (2003).
  11. Moore, M.A.S., Chung, K.Y., Plasilova, M., Schuringa, J.J., Shieh, J.H., Zhou, P. & Morrone, G. Nup98 dysregulation in myeloid leukemogenesis. Ann. N.Y. Acad. Sci. 1106, 114-142 (2007).
  12. Kodiha, M., Chu, A. Lazrak, O. & Stochaj, U. Stress inhibits the nucleocytoplasmic shuttling of heat shock protein hsc70. Am. J. Physiol. 289, 1034-1041 (2005).
  13. Kodiha, M., Brown, C.M. & Stochaj, U. Analysis of signaling events by combining high throughput screening technology with computer based image analysis. Sci. Signal. 1, p12 (2008).

Acknowledgements

This research was supported by grants from CIHR, NSERC and HSFQ to US. MK is a recipient of a fellowship from the Heart and Stroke Foundation of Canada.

Figures

Figure 1. : Comparison of Nup98 staining with different immunofluorescence options.

Fig 1

Immunolocalization of Nup98 in HeLa cells. DNA was visualized with DAPI. Preferential staining of nucleoli (Option 1), nuclear bodies, nucleoli and the cytoplasm (Options 2 and 3) or the cytoplasm (Option 4) was observed.

Author information

Mohamed Kodiha, Rehan Umar & Ursula Stochaj, McGill University

Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.16. Originally published online 22 January 2009.

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