Immunofluorescence for Detection of TOR Kinase Activity In Situ in Photosynthetic Organisms

Materials and reagents

Biological materials

1. Arabidopsis thaliana Columbia ecotype (Col-0) wild-type (WT), young seedlings (5 days old)

2. Chlamydomonas reinhardtii CC125 (137c, mt + nit1 nit2), 15 mL of OD_750nm: 0.6–0.9

1. Double-distilled water (ddH₂O)

2. Sodium phosphate dibasic (Na₂HPO₄) (Sigma, catalog number: 71496)

3. Potassium dihydrogen phosphate (KH₂PO₄) (Sigma, catalog number: 60220-M)

4. Sodium chloride (NaCl) (Anedra, catalog number: AN00716909)

5. Orthophosphoric acid (H₃PO₄) (Sigma, catalog number: 695017)

6. Paraformaldehyde (Agar Scientific, catalog number: R1018)

7. Sodium hydroxide (NaOH) (Fluka, catalog number: 71690)

8. Bovine serum albumin (BSA) 10 mg/mL (Promega, catalog number: R396D)

9. Triton X-100 (Sigma, catalog number: 9036-19-5)

10. Primary polyclonal antibody p-p70 S6K1 α Thr 389 (Santa Cruz, catalog number: sc-11759)

11. Secondary antibody anti-IgG-Alexa 488 (Invitrogen, catalog number: A11008)

12. Propidium iodide (PI) (Invitrogen, catalog number: P1304MP)

13. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, catalog number: D9564)

1. Paraformaldehyde (PFA) 4% (see Recipes)

2. Phosphate buffered saline (PBS) 5× (see Recipes)

3. Permeabilization and blocking buffer (PB) (see Recipes)

1. PFA 4%

   1. Weigh 4 g of PFA and place in 35 mL of double-distilled water at 60 °C, add 30–50 μL of 10 N NaOH, and leave to stir on a hot plate (until the PFA dissolves well).

   2. Add 20 mL of 5× PBS.

   3. Check pH; it should be between 7.2 and 7.8.

   4. Bring up to 100 mL with ddH₂O.

   5. Split into Falcon tubes and store at -20 °C for up to 3 months and protected from light.

   Caution: Highly toxic. Wear protective gloves and work in the fume hood when handling the powder.




2. PBS (5×) (for 1 L)

   Dissolve 3.62 g of Na₂HPO₄ (FW 141.96), 1.05 g of KH₂PO₄ (FW 136.1), and 38.25 g of NaCl (FW 58.44) in 800 mL of ddH₂O. Adjust the pH to 7.4 with H₃PO₄ and then add ddH₂O to the final volume. Dispense the solution into aliquots and sterilize them by autoclaving. The work solution used is 1× (final concentration 8 mM Na₂HPO₄, 2 mM KH₂PO₄, and 140 mM NaCl).




3. Permeabilization and blocking buffer (PB) (for 1.5 mL of PB)

   Add 150 μL of 1% BSA, 45 μL of 10% Triton X-100, and 1,305 μL of 1× PBS (final concentration 1× PBS, 0.1% BSA, and 0.3% Triton X-100).

Equipment

1. Confocal microscope (e.g., Nikon, model: Eclipse C1 Plus)

2. Vacuum pump with a desiccator

Software and datasets

1. Confocal microscope software (e.g., EZ-C1 software Nikon)

2. NIH ImageJ software 1.47 for Windows (https://imagej.net/ij/)

Procedure

1. Tissue fixation (duration 4–6 h)

   1. Remove Arabidopsis or Chlamydomonas samples from the growth medium and wash with 1× PBS.

   2. Add 200 μL of 4% PFA to each tube (must cover the biological material).

   3. Sample incubation

      1. For algae: Incubate for 6 h at 4 °C.

      2. For plant: Cut the root, place it in a microcentrifuge tube, and incubate the tissue in a vacuum desiccator for 6 h at room temperature (approximately 20 °C) to ensure proper tissue penetration.

      Note: Leaves could also be used for the analysis (after removal of chlorophyll and other pigments).




2. Permeabilization and blocking (duration 24 h)

   1. Remove the PFA solution and wash the material with 1× PBS at RT.

   2. Add ~70 μL of PB to each tube (must cover the biological material) and incubate for 24 h at 4 °C.




3. Primary antibody incubation (duration 4 d)

   1. Remove PB from all tubes (except the negative control, in which you should change the PB solution).

   2. Add the antibody at a dilution of 1/50 in PB to a final volume of 50 μL per tube (except the negative control, in which you should change the PB solution).

   3. Incubate for 4 days at 4 °C (move the solution every 1 or 2 days).

   4. Wash the material with 1× PBS (wash, remove, and add back).

   5. Incubate for 24 h at 4 °C.

   Note: In the case of plants, primary antibody can be recovered for another use.




4. Secondary antibody incubation (duration 2 d)

   1. Remove the PBS solution.

   2. Add the anti-IgG-Alexa 488 antibody at a dilution of 1/250 in PB to a final volume of 50 μL per tube.

   3. Incubate for 24 h at 4 °C.

   4. Wash the material with 1× PBS (wash, remove, and add back).

   5. Incubate for 24 h at 4 °C.

   Note: Always protect the samples from light.




5. Nucleus staining (optional)

   Nucleus labeling can be useful to localize the protein of interest at subcellular, cellular, and tissue level.

   
   

   For propidium iodide (PI) staining:

   1. Remove the PBS solution.

   2. Add PI fluorophore to the samples at a dilution of 1/1,000 in 1× PBS to a final volume of 50 μL per tube (protect from light).

   3. Incubate for 5 min at room temperature.

   4. Wash the material four times with 1× PBS (wash, remove, and add back).

   5. Analyze under a confocal microscope.

   
   

   For DAPI staining:

   1. Remove the PBS solution.

   2. Add DAPI fluorophore to the samples at a dilution of 1/1,000 in 1× PBS to a final volume of 50 μL per tube (protect from light).

   3. Incubate for 30 min for Arabidopsis or 5 min for Chlamydomonas at room temperature.

   4. Wash the material three times with 1× PBS (wash, remove, and add back).

   5. Analyze under a confocal microscope.

Data analysis

Sample images were obtained with C1 confocal laser using the following settings: green fluorescence intensity; excitation/emission wavelength = 488/561 nm. Visualization was done with the Super Fluor 40.0×/1.30/0.22 oil spring–loaded objective, and images were processed with the NIH ImageJ software. The positive signal appeared as bright spots in immunolocalization using a P-S6K antibody as was previously reported in rat and mouse tissues and human cell lines [7]. For Arabidopsis, at least eight roots were visualized (Figures 1A and 2A). For Chlamydomonas, eight coverslips were taken from each biological replicate (Figure 3A).

Figure 1. Immunolocalization of P-S6K in the root tip of five-day-old Arabidopsis seedlings. Confocal images of in situ immunofluorescence assays performed with the primary antibody p-p70 S6 kinase α Thr 389 and the secondary antibody anti-IgG-Alexa 488 (green fluorescence). P-S6K detection in the root tip zone of seedlings grown in control Murashige and Skoog (MS) medium (A) and treated with 2 μM AZD-8055 (TOR inhibitor) (B) for 48 h in darkness. Negative control consisted of the omission of primary antibody (C). Scale bars: 20 μm. Insets: Brightfield photos.

Figure 2. Immunolocalization of P-S6K in the elongation and differentiation zone of the root of five-day-old Arabidopsis seedlings. Confocal images of in situ immunofluorescence assays performed with the antibody p-p70 S6 kinase α Thr 389 and the secondary antibody anti-IgG-Alexa 488 (green fluorescence). P-S6K detection in the elongation and differentiation zone of the root of seedlings grown in control Murashige and Skoog (MS) medium (A) and treated with 2 μM AZD-8055 inhibitor (B) for 48 h in darkness. Negative control consisted of the omission of primary antibody (C). Scale bars: 50 μm. Insets: Brightfield photos.

Figure 3. Immunolocalization of P-S6K from Chlamydomonas. Confocal images of in situ immunofluorescence assays performed with the antibody p-p70 S6 kinase α Thr 389 and the secondary antibody anti-IgG-Alexa 488 (green fluorescence). P-S6K detection in Chlamydomonas grown in control medium TAP (A) and treated with 700 nM AZD-8055 inhibitor (B) for 24 h. Negative control consisted of the omission of primary antibody (C). Scale bars: 10 μm. Insets: Brightfield photos.

Validation of protocol

To validate the observed fluorescence pattern of the protein under study, each immunolocalization experiment must include the correct controls. In our case, we used an ATP-competitive TOR inhibitor (AZD-8055 Cayman, catalog number: 16978) to evaluate the specificity of the protocol. The AZD treatment considerably reduced the fluorescence intensity in both Arabidopsis and Chlamydomonas samples, corroborating that the signal detected is specifically due to the TOR activity (Figures 1B, 2B, and 3B). Besides, a sample without the addition of the primary antibody was used to determine the amount of background signal caused by the secondary antibody (Figures 1C, 2C, and 3C). This negative control did not show fluorescence, demonstrating that the technique has a low background. We also corroborated this methodology in other plant tissues such as lateral and hair roots (Figures S1 and S2).

General notes and troubleshooting

Troubleshooting

Regarding plant samples, the main limitation of this protocol is its restricted applicability to young and permeable tissues. To ensure the fixation of the Arabidopsis samples, it is mandatory to make a cut in the fresh tissue, and it is critical to perform the vacuum step, also for the root tip. While this step is not essential for other plant tissues and algae samples, it improves the methodology. In the case of cotyledons and young leaves, special care must be taken with interferences caused by chlorophyll, which must be removed using solvents. For microalgae, the material quantity is a crucial factor (~2–4 mg dry weight): it must be enough to be processed (taking into account material losses due to washing) and not be in excess to avoid interferences with cell fixation and permeabilization.

Acknowledgments

This work was supported by the National Agency for Promotion of Science and Technology (ANPCyT, PICT2019-2118), National Scientific and Technical Research Council (CONICET, PIP-11220200101701CO) and National University of Mar del Plata (UNMdP, EXA1142/23). This protocol was adapted and modified from Loos et al. [8] (doi: 10.1128/AAC.01808-19, PMID: 32540980).

Competing interests

The authors declare that they have no competing interests.

References

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Supplementary information

The following supporting information can be downloaded here:

1. Figure S1. Immunolocalization of P-S6K in the root tip of five-day-old Arabidopsis seedlings
2. Figure S2. Immunolocalization of P-S6K in the root of five-day-old Arabidopsis seedlings