Lsm 780 nlo inverted axio observer z1 confocal microscope

ExAdEx samples were fixed with 4% PAF and then incubated with primary anti-collagen 1 (Abcam ab260043), anti–elastin (Abcam ab21610), anti-laminin (ab11575), anti-fibronectin Santa Cruz sc8422) and anti-CD31 (Abcam ab28364) antibodies overnight at 4 °C and then with the corresponding secondary antibody for 45 min at room temperature. Lipid droplets were stained with Oil Red O (ORO), and nuclei were stained with DAPI. Samples were visualized on an LSM 780 NLO inverted Axio Observer Z1 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using a Plan Apo 25× multi immersion (oil, glycerol, water) NA 0.8 objective. The SHG light source was a Mai Tai DeepSee (Newport Corp., Irvine, CA, USA) tuned to 880 nm. A forward SHG signal was detected with an oil condenser (1.4 NA), bandpass filter 440/40 nm and transmission PMT. Backward SHG was collected with a GaAsP (BIG) nondescanned module of 440/10 nm.

Gonad samples were fixed with 4% paraformaldehyde overnight, processed for paraffin embedding, and sectioned at 5 μm thick. The following dilutions of primary antibodies were used: AMH/MIS (c-20, sc-6886, Santa Cruz), 1:200; DMRT1 (HPA027850, Sigma), 1:100; FOXL2 (NB100-1277, Novus), 1:200; GATA1 (N6, sc-265, Santa Cruz), 1:200; GATA4 (C20, sc-1237, Santa Cruz), 1:200; 3βHSD (P18, sc-30820, Santa Cruz), 1:200; P27 (Kip1, sc-528, Santa Cruz), 1:200; LAMA1 (L9393, Sigma), 1:150; SF1 (kindly provided by Ken Morohashi), 1:1000; SOX8 (kindly provided by Elisabeth Sock [Stolt et al., 2005 (link)]), 1:1000; SOX9 (HPA001758, Sigma), 1:200; and TRA98 (ab82527, Abcam), 1:200. Counterstain with DAPI was used to detect nuclei. Immunofluorescence of secondary antibodies were detected with an Axio ImagerZ1 microscope (Zeiss) coupled to an Axiocam mrm camera (Zeiss) or a LSM 780 NLO inverted Axio Observer.Z1 confocal microscope (Carl Zeiss Microscopy GmbH, Jena,Germany) using a Plan Apo 10X dry NA 0.45 objective. Images were processed with Axiovision LE and Serif Affinity Photo software. Immunostaining experiments were performed on gonads from at least three mice for each genotype.