Cy3 donkey anti rabbit igg

Twenty-four hours after OGD, imunofluorescence staining was performed to evaluate the DCC expression. Neurons were washed with PBS for three times and then fixed in 4% paraformaldehyde (pH 7.4) for 15 min. Cells were incubated at 4°C overnight with rabbit anti-DCC antibody (1:20, Abcam, UK). After three washes with PBS, they were further incubated with corresponding secondary antibody, Cy3 donkey anti-rabbit IgG (1:400, Jackson Immunoresearch, USA) at room temperature for 2 h. The nuclei were stained with DAPI (5 μg/ml; Beyotime, USA). Glass slides were viewed under a ZEISS LSM 780 confocal microscope (Carl Zeiss, Germany), and the OD was quantified with ImageJ software as described previously. All trials were repeated three times.

Samples were washed three times with 1×PBS and then fixed for 10 min with 4 % paraformaldehyde (PFA) (Merck, Darmstadt, Germany)/1×PBS. After being washed with 1×PBS three times for 10 min each, the samples were treated with a blocking solution (10 % normal donkey serum/1×PBS) (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature (RT). The samples were subjected to consecutive treatments with primary and secondary antibodies for 1 h each at RT. The samples were treated with DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories, Peterborough, UK) for 5 min after the secondary antibody treatments.
The primary antibodies used in this study are shown in Additional file 1. The secondary antibodies used in this study include an Alexa Fluor 594 goat anti-mouse IgG (1:500), fluorescein isothiocyanate (FITC) donkey anti-mouse IgG (1:500), Cy3 donkey anti-rabbit IgG (1:500), FITC donkey anti-rabbit IgG (1:500), and FITC donkey anti-chicken IgY (1:500) (all from Jackson ImmunoResearch).

Kidneys were fixed in 4% PFA overnight at 4 °C, washed with PBS, embedded in paraffin blocks, 5 µm sections were generated and blocked in 10% normal donkey serum in PBS containing 0.3% Triton-X (blocking buffer) at room temperature for 1 h. Primary antibodies used included chick anti-GFP (1:250; Aves Labs), and rabbit anti-phospho-S6 ribosomal protein Ser240/244 (1:500; Cell Signaling Technology, Danvers, MA, USA, Catalog #5364). Slides were incubated with primary antibody in blocking buffer overnight at 4 °C, and washed 3 times in PBS with 0.1% Tween-20 for 20 min at room temperature. After washing, slides were incubated with secondary antibody (Alexa Fluor 488 donkey anti-chicken; 1:250; Jackson ImmunoResearch, Catalog #703-545-155 and Cy3 donkey anti-rabbit IgG; 1:250; Jackson ImmunoResearch) at room temperature for 1 h followed by washing and DAPI staining at 4 °C. Slides were mounted in Prolong Gold Antifade Reagent (Cell Signaling Technology) and imaged with a confocal Nikon A1R inverted microscope with a 20X objective lens using a pinhole of 1.2 μm on all channels at a resolution of 1024 × 1024.

The microtome sections were mounted, deparaffinized, and rehydrated. The slices were then incubated in a moist chamber for 48 h at 4°C with one or two primary antibodies (Anti-Neurophysin 2/NP-AVP mouse IgG, 1:500, Millipore, USA, Anti-OT rabbit IgG, 1:4000, produced by G. Alonso, Montpellier, France) (Alonso et al. 2005 (link)).
After rinsing in PBS, sections were incubated for 1 h at room temperature with the corresponding secondary antibodies conjugated with Cy3 (donkey anti-rabbit IgG, Jackson ImmunoResearchLaboratories, USA, 1:2000) or Alexa Fluor 488 (goat anti-mouse IgG, Invitrogen Molecular Probes, USA, 1:2000). The antibodies were diluted in PBS containing 2% Bovine Serum Albumin (BSA) and 0.1% Triton X-100. After rinsing in PBS, sections were incubated for 30 min in DAPI (1:1000) (4′,6-diamidino-2-phenylindole) a nuclear counterstain for fluorescence microscopy.
Labeled sections were rinsed in PBS, mounted in Mowiol, and observed under Zeiss Axio Imager 2 fluorescence microscope with Apotome (IGF Montpellier, France).
The specificity of the vasopressin, oxytocin and commercial antibodies has been assessed by absorption tests. Additional negative and positive controls were applied. This allowed us to confirm the validity of the staining pattern and to exclude experimental artifacts.

Sections were washed in TBS, incubated in blocking solution (TBS containing 0.1% Triton X-100, TBS-T, and 5% donkey serum) 2 h at RT, incubated 72 h at 4°C in blocking solution supplemented with c-Fos (1:500; rabbit polyclonal #sc-52, Santa Cruz Biotechnology) and kisspeptin [1:10,000; sheep polyclonal #AC053, generous gift of I. Franceschini (Franceschini et al., 2013 (link))] primary antibodies. Sections were then washed in TBS and incubated in TBS-T supplemented with secondary antibodies (1:500; Cy3 donkey anti-rabbit-IgG and Alexa-488 donkey anti-sheep-IgG, Jackson ImmunoResearch) for 2 h at RT. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml, Sigma-Aldrich) for 2 min. Slides were mounted with Fluoromount^TM (Sigma-Aldrich).