Cy3 conjugated secondary antibody

Cochleae of hearing Cav1.3DCRD^HA/HA mice and WT littermates (aged 3–11 weeks) were fixed by injection of Zamboni’s fixative into the round and oval window and incubation for 8 min on ice, followed by rinsing with PBS. The organ of Corti was dissected and mounted on a slide using CellTak (BD Bioscience). Whole-mounts were stained using the following solutions: PBS, blocking buffer (1% BSA in PBS), permeabilization buffer (0.5% Triton X-100 in PBS), reaction buffer (0.5% BSA, 0.2% Triton X-100 in PBS), washing buffer (0.1% Triton X-100 in PBS). Whole-mounts were embedded with Vectashield mounting medium with DAPI (Vector UK) and viewed using a confocal Zeiss LSM 700. Whole-mounts were double-labeled by simultaneous incubation of an Alexa488-conjugated anti-HA antibody and antibodies directed against Cav1.3, CtBP2/RIBEYE or Cavβ2 (see above), which were detected using a Cy3-conjugated secondary antibody (Jackson Immunoresearch).

Brains from 1-month-old mice were perfused with phosphate-buffered 4% paraformaldehyde (PFA) followed by extraction of the brain, embedded in OTC freezing solution and sectioned sequentially from the olfactory bulb lobule towards the cerebellum. Cross-sections were labelled with primary antibodies followed by incubation with Cy3-conjugated secondary antibody (Jackson ImmunoResearch) and Alexa Fluor 488 anti-mouse IgG (Invitrogen). Culture cells were fixed in 3% paraformaldehyde and immunostained with primary antibodies. Cy3 anti-rabbit IgG (Jackson Laboratories) and Alexa Fluor 488 anti-mouse IgG (Invitrogen) were used as secondary antibodies before confocal microscopy imaging. Images were acquired on a Nikon C1si confocal microscope, with a Plan Apo × 40, numerical aperture (NA) 1.3 and/or Plan Apo × 60, NA 1.45 objective (Melville, NY).

Tumors and lungs were embedded in OCT and subsequently sectioned. Lungs were stained with H&E in order to detect metastatic lesions. Tumor sections (10 μm) were analyzed for microvessel density (MVD) by immunostaining using an anti-CD31 antibody (1:200, BD Biosciences, San Jose, CA, USA) followed by a Cy3-conjugated secondary antibody (1:500, (Jackson). Vessel structures per field were counted and plotted. At least 5 fields per tumor, normally n > 15 fields per group, were counted. Cancer survey tissue microarrays (TMA, OriGene Technologies, Inc., Rockville, MD USA) or tumor sections from human breast (n = 15) and colon (n = 15) carcinoma samples obtained from the Department of Pathology at the Rambam Medical Center (Haifa, Israel), were immunostained with human IL31RA antibody (1:100, Abcam) followed by histidine peroxidase anti-mouse and anti-rabbit secondary antibodies (Nichirei, Japan). Staining was developed using AEC simple stain solution (Nichirei). Hematoxylin (Sigma) was used as a counterstain. The samples were analyzed by a pathologist for positive staining. All human studies were approved by the ethic committee at the Rambam Medical Center after patients signed an informed consent.

HKC‐8 cells were cultured on coverslips and fixed with 4% paraformaldehyde for 15 min at room temperature and immersed in 0.2% Triton X‐100 for 10 min. After blocking with 10% donkey serum for 30 min, the cells were immunostained with primary antibodies against fibronectin (F3648; Sigma‐Aldrich, St. Louis, MO) and a Cy3‐conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Photos were taken with fluorescence microscopy (Leica DMi8; Leica Microsystems, Buffalo Grove, IL).

Serial hippocampal or hypothalamus sections (40 μm) were made on an oscillating tissue slicer. For c-FOS and CRF immunofluorescence, the sections were incubated with rabbit anti-c-FOS antibody (1:400; Synaptic Systems) and anti-CRF (1:200; Santa Cruz Biotechnology) in 0.1 M PBS with 3% goat serum and 0.3% Triton X-100, and binding was visualized with a Cy3-conjugated secondary antibody (1:200; Jackson immunoresearch). Images of immunostained neurons in all groups were captured with a Zeiss Axio Cam MRC 5(D) camera mounted on Carl Zeiss Axio Observer A1 microscope under same conditions. An experimenter coded all slides from the experiments before quantitative analysis. All CRF-positive cells and c-FOS-positive in the PVN were counted in each section by another experimenter blinded to the study code.

Brains were removed and cryoprotected in 30% sucrose after trans‐cardiac perfusion with 4% paraformaldehyde. Samples were cut into coronal free‐floating sections (25 μm). For immunofluorescence staining, sections were blocked and incubated overnight at 4 °C with AT8 (Thermo Fisher Scientific, Carlsbad, CA, USA, #MN1020, 1:25). Cy3‐conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA, 715‐165‐150, 1:200) was incubated 1 h at RT, and DAPI (Sigma Chemical Aldrich, Milwaukee, WI, USA) was used to stain nuclei. Controls included: Tau KO brains stained with AT8, and sections treated with secondary antibody alone. Neither showed appreciable staining. Images were acquired using Leica TCS SP5 confocal laser scanning microscopes (Leica, Richmond, IL, USA). The percentage of the overall AT8‐positive cells in the CA1 areas of hippocampus was quantified using the imagej NIH software for Windows (Bethesda, MA, USA).