This protocol was developed by Frank Wippich and was used in Wippich, F., et al. (2013) Cell152(4), 791–805.

  1. Culture mammalian tissue cells overnight in complete medium (DMEM with Glutamax and 10% FCS) on coverslips, other carriers, or in a multi-well plate suitable for fluorescence microscopy imaging.
  2. Serum-starve cells for 14 hours in DMEM without serum.
  3. Treat cells for 45 min with 0.5 mM Arsenite in DMEM to induce the formation of Stress Granules (SGs).
  4. Wash cells twice with PBS and fix with 4% paraformaldehyde (PFA) in PBS.
  5. Permeabilise cells for 10 min with 0.1% Triton X100 in PBS
  6. Block a-specific binding sites with 1% BSA in PBS for 30 min
  7. Stain SGs using a mouse monoclonal antibody against PABP1 (10E10) from Santa Cruz Biotechnology (sc-32318), diluted 1:500 in PBS containing 1%BSA
  8. Stain for mTOR using a rabbit polyclonal antibody against the N-terminus of human FRAP1  (N-19)-R from Santa Cruz Biotechnology (sc-1549-R), diluted 1:500 in PBS containing 1%BSA.
  9. !!! NOTE !!! Different mTOR antibodies, e.g. some from Cell Signaling, do not efficiently stain the Stress Granule pool of mTOR, but rather the late endosome/lysosomal pool of mTOR. This could be due to specific epitopes on mTOR that are differently accessible by different antibodies depending on the subcellular localisation and/or active conformation of mTOR. This staining should therefore always be validated using a tagged construct expressed in cells and immunofluorescent staining against this tag, for instance using myc-tagged Raptor (as done in Wippich, F., et al. (2013) Cell152(4), 791–805).
  10. Incubate with primary antibodies for 1 hour, wash 3 times extensively with PBS containing 1%BSA.
  11. Incubate with Alexa Fluor-labelled goat anti-mouse and goat anti-rabbit secondary antibodies (we routinely use Alexa Fluor 488- and Alexa Fluor 568-labelled secondary antibodies), diluted 1:500 in PBS with 1%BSA.
  12. Wash extensively with PBS with 1%BSA (3x 10 min).
  13. Stain nuclei of cells with DAPI (1:1000 in PBS from a stock solution of 100 µg/ml) for 5 min at RT.
  14. Wash cells with PBS and proceed to mount coverslip on slide (using for instance Immu-Mount) or keep cells in PBS when using a multi-well plate.
  15. Image cells with appropriate microscope (spinning disk or laser scanning confocal microscope allow better imaging of SGs) equipped with appropriate excitation wavelengths and emission filters at at least 40x magnification.
  16. SGs will be visible as bright roundish granules positive for PABP1 scattered in the cytoplasm of cells.