Plan apochromat 40x 1.3 oil dic m27

Cells seeded on top of collagen matrices were fixed with 4% formaldehyde for 15 min at room temperature. Then, cells were permeabilised for 20 min using 0.3% Triton X-100 in 5% BSA-PBS, blocked in 5% BSA-PBS for 30 min and immunostained with primary antibody overnight at 4 °C. Next, samples were incubated with Alexa Fluor^TM 546-phalloidin (A22283, Thermo Fisher, 1:400) and Goat anti-rabbit Alexa Fluor^TM 488 (A-11008, Thermo Fisher, 1:1000) or Goat anti-mouse Alexa Fluor^TM 488 (A-11029, Thermo Fisher, 1:1000) secondary antibodies for 2 h at room temperature. Nuclei were stained with Hoechst prepared in PBS. Antibodies were diluted in 5% BSA-PBS.
Images were taken with a Zeiss LSM 510 Meta confocal microscope with Plan-Apochromat 40x/1.2 NA (water) objective lenses, Zeiss LSM 710 confocal microscope with Plan-Apochromat 40x/1.3 Oil DIC M27 and Zeiss LSM 880 confocal microscope with Airyscan superresolution mode and Plan-Apochromat 63x/1.4 Oil DIC M27 objective lenses (Carl Zeiss, Germany). Zen software was used to acquire images (Carl Zeiss, Germany). Images were analysed using ImageJ software (NIH). For pMLC2 quantification, fluorescence signal was quantified by calculating the area occupied by pMLC staining in single cells relative to the cell area. For pMLC2 distribution, line scan analysis was performed in Fiji 1.53t using the Plot profile plug in.

Coronal sections (300 µm) were obtained in the region located between 2.10 mm and 1.54 mm rostral to the bregma. Pyramidal neurons of the layer 2/3 in the prelimbic region of the medial prefrontal cortex were injected with biocytin. Streptavidin-conjugated HRP was used to label the injected neurons using a green fluorescent substrate. Each section was then mounted on a slide and confocal images were obtained using at The Light Microscopy Facility, the Max Planck Florida Institute, using a confocal microscope (LSM 780; Carl Zeiss; Plan Apochromat 40x/1.3 Oil DIC M27) at room temperature. Image acquisition and processing were accomplished using ZEN 2011 (64 bit) software (Carl Zeiss, Oberkochen, Germany) with only minor manipulations of the images setting the fluorescence intensity in non-saturating conditions and maintaining similar parameters for each acquired image. Pictures were elaborated and spine density calculated using ImageJ and Neurolucida (MicroBrightField, Williston, VT) software. Only apical dendrites of second order were analyzed. At least three segments/neuron (>40 µm; average of 420 µm/mouse) with similar diameter (average of 1.06 ± 0.21 µm) that did not show intersections with other dendrites were considered for analysis. All measurements were performed by an experimenter blind to the mice genotype.