PCC7120 with fluorophore-conjugated antibodies by fluorescence microscopy. Nail varnish Main polyclonal antibody against All2320 peptide ( Mandakovic sp. PCC7120 is usually produced axenically in BG-11 liquid medium at 24 C under white light (25 mol m-2 sec-1) and shaking at 90 rpm. Fixation and permeabilization 50 l 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. Fix the cell spots in 70% ethanol and incubate for 30 min at -20 C. The slide is usually immersed in chilly 70% ethanol contained in a Petri dish. The slides are air-dried for 20 min at room temperature. Make use of a hydrophobic PAP pen to draw a circle round the slide-mounted cell spot and let it dry for 15 min at room temperature. Labeling 10Z-Hymenialdisine process 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 10Z-Hymenialdisine the drop each time with a pipette. Incubate with a drop of 3% BSA, 0.2% Triton X-100 in PBS for 1 h at 4 C in a moisture chamber Rabbit Polyclonal to Stefin A and remove this blocking answer. 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. Wash with 0.05% Triton X-100 in PBS for 2 min at room temperature, and repeat three times. 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 g/ml) for 45 min at 4 C, in a moisture chamber. Wash with 0.05% Triton X-100 in PBS for 2 min at room temperature for three times. Add a drop of Prolong Antifade reagent to the sample slide, 10Z-Hymenialdisine and then cover this with a cover slip while taking care not to create air 10Z-Hymenialdisine flow bubbles. Seal with nail varnish. The slides are visualized with a Fluoview FV1000 Confocal Microscope and images are acquired in 16 bits. Alexa Fluor 488 is usually excited at a wavelength of 495 nm and emission is usually measured at 509 nm. To visualize autofluorescence due to phycobilisomes, samples are excited using 565 nm and fluorescence emission is usually monitored at 590 nm (Physique 1). Open in a separate window Physique 1. Immunolocalization of CyDiv in sp. PCC7120. Deconvoluted image of a Z-stack. A. Autofluorescence; B. Image signal derived from main 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 m. Data analysis Images of Z-stacks were processed using ImageJ software ( Schneider em et al. /em , 2012 ). For each channel of images, the point-spread function (PSF) was calculated using the Given birth to and Wolf model within the PSF Generator plugin ( Kirshner em et al. /em , 2013 ). Image deconvolution was performed with the Deconvolution Lab plugin 10Z-Hymenialdisine with Richardson-Lucy algorithm using 10 iterations (Vonesch and Unser, 2008). Quality recipes PBS buffer (pH 7.4) 137 mM NaCl 2.7 mM KCl 1.4 mM Na2HPO4 1.4 mM KH2PO4 em Note: The PBS is filtered through a filter with a pore size of 0.2 m and stored at room heat. /em Acknowledgments The protocol described has been altered from ( Plominsky em et al. /em , 2013 ; Miyagishima em et al. /em , 2014 ). This work was supported by Fondecyt grants #1131037, 1161232 and Fellowships for Graduate Student of Chilean Government # 21100780 and 21150983. Citation Readers should cite both the Bio-protocol article and the original research article where this protocol was used..