Donkey anti rabbit igg

Immunocytochemistry was performed in cultured organs of Corti using antibodies (Table S1) against anti-VGLUT3 (1:500, Synaptic Systems, Göttingen, Allemagne, #135204 RRID:AB_2619825) to label IHCs and anti-NF200 (1:400, Sigma-Aldrich, Merck group, Saint Quentin Fallavier, France, #N0142 RRID:AB_477257) to label the SGNs and their peripheral axons. All secondary antibodies were used at a dilution of 1:1000. These included goat anti-guinea pig and donkey anti-rabbit IgG conjugated to Alexa 488 and Alexa 594, respectively (Molecular Probes, Illkirch, France, #A-11073 RRID: AB_2534117, #A-21207, RRID:AB_141637). DNA was stained using Hoechst 33342 (1:5000, Thermo Fisher Scientific group Illkirch, France, #62249). Fluorescent tags were visualized using confocal microscopy (Zeiss 880 Airyscan). The quantitative analyses of NF200-labeled fibers (6 cochleae per condition and per time point) were carried out in the cochlear regions coding 32 kHz, each containing 10–15 hair cells. The terminals are quantified as the average number of NF200-labeled fibers in contact with an IHC.

Worms were dissected and processed as described before (14 (link),31 (link)) and treated with respective primary antibody overnight and secondary antibody for 4 h at room temperature. Rabbit anti-H3K4me3 (1:1000) was purchased from Abcam (ab8580). Mouse monoclonal antibody against H3K4me2 (CMA303; 1:20) were gifts from Dr. Hiroshi Kimura (Osaka University, Japan). Mouse monoclonal antibody against GFP (1:500) was purchased from Millipore. Rabbit anti-TRA-1 (1:100) antibodies were generous gifts from Drs. David Zarkower (University of Minnesota) and Andrew Spence (University Toronto). 4',6-Diamidino-2-Phenylindole (DAPI) (Sigma, 2 μg/μl) was used to counter-stain deoxyribonucleic acid (DNA). All secondary antibodies were purchased from Molecular Probes and were used at 1:500 dilutions: goat anti-mouse IgG (Alexafluor 488); goat anti-rabbit IgG (Alexafluor 594), donkey anti-rabbit IgG (Alexafluor 488); donkey anti-mouse IgG (Alexafluor 594). Worms were mounted in anti-fade reagent (Prolong Gold, Molecular Probes). Images were collected using a Leica DMRXA fluorescence microscope and analyzed with Simple PCI software (Hamamatsu Photonics).

Larval fish were fixed overnight at 4 °C in 4% paraformaldehyde/1xPBS, after which they were dehydrated through an ethanol series and stored at −20 °C until use.
In situ hybridizations were performed as previously described25 (link). hcrt, npvf and gpr147 ORFs was cloned in a pCS2+ vector using zebrafish cDNA and antisense DIG labelled probes were transcribed using the linearized pCS2+ plasmid containing the ORF. In situs were visualized with Fast Red (Roche) as substrates.
Immunohistochemical stainings were performed as previously described26 (link), using either anti-GFP (1/1000, Torrey Pines Biolabs), anti-mCherry (1/200, Abcam), anti-5HT (1/1000–1/200, ImmunoStar) and anti-NPVF (1/200, Abcam) as primary antibodies and Alexa 488, Alexa 555, Alexa 594, or Alexa 657-conjugated goat anti-rabbit IgG, goat anti-mouse IgG, or donkey anti-rabbit IgG (1/1000–1/200) as secondary antibodies (Molecular Probes).

Cryopreserved PBMCs were used to evaluate the H3Ac in lymphocytes by flow cytometry as described previously¹³ (link). Briefly, after a 20 min blocking with PBS 10% FBS, cells were stained for 30 min with anti-acetyl histone H3 polyclonal rabbit (MerckMillipore 06–599) or with the control stain using normal rabbit serum (LifeTechnologies 10510). After washing, a 30 min incubation with the secondary antibody in the dark was performed (donkey anti-rabbit IgG, LifeTechnologies A21206). The median fluorescence intensity (MFI) for acetyl histone H3 stain was determined and the background was subtracted using the MFI of the isotype control staining.

For immunofluorescence assays, cells were fixed by incubation in 4% paraformaldehyde for 30 minutes, permeabilized by incubation in 0.2% Triton X-100 for 30 minutes and blocked by incubation with 3% BSA and 5% donkey serum in PBS (Chemicon). The cells were then incubated overnight with antibodies directed against OCT3/4 (1/100) (Abcam ab19857), SSEA-4-AF555 (1:50) (BD 560218) and Tra-1-60-AF488 (1:100) (Miltenyi Biotec). The cells were washed and Alexa Fluor 555-conjugated (1:500) donkey anti-rabbit IgG (Life Technologies) was added for the detection of Oct3/4.