Materials and Reagents

Pipette tips
1.5 ml tubes (Eppendorf)
50 ml tubes (Falcon tubes)
Poly-L-lysine coated glass slides (Sigma-Aldrich, catalog number: P0425-72EA )
Cover slips
Petri dish
Filter with a pore size of 0.2 µm
Filamentous cyanobacterium, Anabaena sp. PCC7120
BG-11 liquid supplied with 10 mM NaHCO₃ (Rippka, 1988)
Sodium hydrogen carbonate (NaHCO₃) (EMD Millipore, catalog number: 106329 )
Ethanol (EMD Millipore, catalog number: 1.00983.2500 )
Triton X-100 (Winkler Limitada, catalog number: BM-2020 )
Bovine serum albumin (BSA) (Divbio Science, catalog number: 41-903-100 )
Tween-20 (Winkler Limitada, catalog number: TW-1652 )
Secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific, Invitrogen, catalog number: A11008 )
ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Invitrogen^TM, catalog number: P36930 )
Nail varnish
Primary polyclonal antibody against All2320 peptide (Mandakovic et al., 2016)
Sodium chloride (NaCl) (EMD Millipore, catalog number: 106404 )
Potassium chloride (KCl) (EMD Millipore, catalog number: 104938 )
Sodium dihydrogen phosphate (Na₂HPO₄) (EMD Millipore, catalog number: 106559 )
Potassium phosphate monobasic (KH₂PO₄) (EMD Millipore, catalog number: 529568 )
PBS buffer (pH 7.4) (see Recipes)

Equipment

Pipettes
Hydrophobic PAP pen (Thermo Fisher Scientific, catalog number: 008877 )
Freezer at -20 °C
Incubator at 4 °C
Incubator at 55 °C
Incubator at 24 °C with white light
Olympus Fluoview FV1000 Confocal Microscope (Olympus, model: Fluoview^TM FV1000 ) and objectives of 60x/1.35 NA oil immersion and 100x/1.40 NA oil immersion. Laserline Argon 488 (Excitation 495 nm, Emission 509 nm) and Laserline DPSS (Excitation 565 nm, Emission 590 nm)
Moisture chamber (A dark plastic box with a moistened paper inside, PolarSafe^TM Polypropylene Freezer Storage Box) (Argos Technologies, catalog number: R3130 )

Software

ImageJ software (https://imagej.net)

Procedure

Organism and growth conditions
1. Anabaena sp. PCC7120 is grown axenically in BG-11 liquid medium at 24 °C under white light (25 µmol m^-2 sec^-1) and shaking at 90 rpm.
Fixation and permeabilization
1. 50 µl of cyanobacterial culture (OD₇₅₀ = 0.3) is added to a poly-lysine microscopy slide and dried for 20 min at 55 °C. Do not fix the cells with organic solvents or aldehydes.
2. Fix the cell spots in 70% ethanol and incubate for 30 min at -20 °C. The slide is immersed in cold 70% ethanol contained in a Petri dish.
3. The slides are air-dried for 20 min at room temperature.
4. Use a hydrophobic PAP pen to draw a circle around the slide-mounted cell spot and let it dry for 15 min at room temperature.
Labeling procedure
1. Permeabilize the cells by adding a drop of 0.05% Triton X-100 in PBS for 2 min at room temperature, and repeat it three times by removing the drop each time with a pipette.
2. Incubate with a drop of 3% BSA, 0.2% Triton X-100 in PBS for 1 h at 4 °C in a moisture chamber and remove this blocking solution.
3. The cells are incubated with the primary antibody diluted 1:100 in a solution with 1% BSA, 0.05% Tween-20 in PBS. Pre-immune serum diluted 1:100 in a solution with 1% BSA, 0.05% Tween-20 in PBS was used as a control to ensure that the primary antibody is working. The cells with the solutions are incubated for 2 h at 4 °C, in a moisture chamber.
4. Wash with 0.05% Triton X-100 in PBS for 2 min at room temperature, and repeat three times.
5. Incubate with secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (diluted in PBS with 1% BSA and 0.05% Tween-20, final concentration 10 µg/ml) for 45 min at 4 °C, in a moisture chamber.
6. Wash with 0.05% Triton X-100 in PBS for 2 min at room temperature for three times.
7. Add a drop of Prolong Antifade reagent to the sample slide, and then cover this with a cover slip while taking care not to create air bubbles. Seal with nail varnish.
8. The slides are visualized with a Fluoview FV1000 Confocal Microscope and images are acquired in 16 bits. Alexa Fluor 488 is excited at a wavelength of 495 nm and emission is measured at 509 nm. To visualize autofluorescence due to phycobilisomes, samples are excited using 565 nm and fluorescence emission is monitored at 590 nm (Figure 1).
  
  Figure 1. Immunolocalization of CyDiv in Anabaena sp. PCC7120. Deconvoluted image of a Z-stack. A. Autofluorescence; B. Image signal derived from primary antibody anti-CyDiv and secondary antibody Alexa Fluor 488 goat anti-rabbit IgG; C. Merged image of the autofluorescence and CyDiv-Alexa Fluor 488 fluorescence. White scale bar = 5 µm.

Data analysis

Images of Z-stacks were processed using ImageJ software (Schneider et al., 2012). For each channel of images, the point-spread function (PSF) was calculated using the Born and Wolf model within the PSF Generator plugin (Kirshner et al., 2013). Image deconvolution was performed with the Deconvolution Lab plugin with Richardson-Lucy algorithm using 10 iterations (Vonesch and Unser, 2008).

Recipes

PBS buffer (pH 7.4)
137 mM NaCl
2.7 mM KCl
1.4 mM Na₂HPO₄
1.4 mM KH₂PO₄
Note: The PBS is filtered through a filter with a pore size of 0.2 µm and stored at room temperature.

Acknowledgments

The protocol described has been modified from (Plominsky et al., 2013; Miyagishima et al., 2014). This work was supported by Fondecyt grants #1131037, 1161232 and Fellowships for Graduate Student of Chilean Government # 21100780 and 21150983.

References

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