Mouse anti huc d

Immunofluorescence was performed as described previously19 (link) using the following primary antibodies: rat anti-BrdU (dividing cell marker, 1:500, Thermo Scientific), mouse anti-Zpr1 (red/green cones, 1:250, ThermoFisher), mouse anti-GS (Müller glia, 1:500, Millipore), and mouse anti-HuC/D (1:300, amacrine and ganglion cells, ThermoFisher). The following secondary antibodies were used: Alexa Flour 488 donkey anti-mouse IgG (1:1000, ThermoFisher), Cy5 donkey anti-mouse (1:1000, ThermoFisher), Alexa Four 488 goat anti-rat IgG (1:1000, ThermoFisher), and Cy3 donkey anti-rat (1:1000, Jackson Immunoresearch). Antigen retrieval for BrdU staining was performed by either boiling the sections in 10 mM sodium citrate for 20 min and cooling for another 20 min or treating the sections with 2 N HCl at 37 °C for 25 min, followed by a rinse with 0.1 M sodium borate solution (pH 8.5) for 5 min.

For the immunohistochemistry analysis, we used the following primary antibodies: rabbit anti-Tamalin (1:200, Novusbio), mouse anti-HuC/D (1:100, Molecular probes), mouse anti-olig2 (1:200, IBL America), and mouse anti-proliferating cell nuclear antigen (PCNA) (1:200, DAKO). To detect fluorescent antibody labeling, we used Alexa 488, 568, 647-conjugated secondary antibodies (1:1000, Molecular Probes). We captured fluorescent pictures of transverse sections using an A1 laser-scanning confocal microscope (Nikon, 1-um z-stack), and wholemount lateral images were obtained using an Eclipse Ti2 Spinning disk confocal microscope (Nikon, 2.5-um z-stack). For in situ RNA hybridization, tamalin and the arf6 open reading frame were cloned into the pGEM T-easy vector (Promega). Not1 and SacII restriction enzyme were used for linearization and transcribed using a DIG labeling combination. Whole-mount or fluorescent in situ RNA hybridization were performed as previously described [36 (link),37 (link)]. The primers were designed using the following sequences:
Tamalin forward: 5′-AGGAGTCCTTTGGCTTCG-3′, Tamalin reverse: 5′-GCTTTCCTCCTCCTCCAGAG-3′, Arf6a forward: 5′-ATTTATGCCCAGCCAAC-3′, and Arf6a reverse: 5′-TTCATTGGCGTTAGGATTTG-3′.

The following antibodies and dilutions were used: mouse anti-acetylated tubulin (Sigma; 1:500), mouse anti-HuC/D (Molecular probes; 1:500), rabbit anti-PH3 (Millipore; 1:500), mouse anti-znp1 (Developmental Studies Hybridoma Bank; 1:100). Primary antibodies were detected with appropriate secondary antibodies conjugated to either Alexa 488 or Alexa 568 (Molecular probes) at a 1:500 dilution. For immunostaining, embryos were fixed in 4% paraformaldehyde 1X PBS overnight at 4 °C, then washed with 1xPBS. Samples were then blocked with 0.5% triton in PBS and 10% sheep serum and then incubated with primary antibody overnight at 4 °C (diluted in PBS + 2% sheep serum). Larvae were then washed with PBS for a few hours and then incubated with secondary antibody in 0.5% triton in PBS and 2% sheep serum overnight at 4 °C. Embryos were incubated for 30 min in PBS triton containing 50 μg/ml of DAPI for nuclei counterstaining when needed. Stained larvae were then imaged with a Leica SP8 confocal microscope.

Cells or tissues were fixed, washed, and permeabelized with 0.1 % Triton X-100 for 30 min and then exposed to the following primary antibodies: mouse anti-Tuj1 (1:100, Covance, Dedham, MA, USA), mouse anti-Hu C/D (1:100, Life Technologies, Carlsbad, CA, USA), rabbit anti-p75 (1:500, Promega, Madison, WI, USA), rabbit anti-Sox2 (1:50, Abcam, Cambridge, MA, USA), rabbit anti-Doublecortin (1:250, Abcam), rabbit anti-Olig2 (1:250, Millipore, Billerica, MA), goat anti-GFAP (1:500, Abcam), goat anti-GFP (1:400, Rockland, Limerick, PA, USA). Secondary antibodies included goat anti-mouse IgG Alexa Fluor 546, goat anti-rabbit Alexa Fluor 488, donkey anti-goat Alexa Fluor 488, donkey anti-goat Alexa Fluor 546, donkey anti-mouse Alexa Fluor 546, donkey anti-mouse Alexa Fluor 488, all from Life Technologies (Carlsbad, CA, USA). Cell nuclei were stained with DAPI (Vector Labs, Burlingame, CA, USA).

GLP-1 inputs to CRH and Oxytocin neurons in the PVH were visualized in GCG-Cre::SynTom::Lep^ob/ob mice and GCG-Cre::SynTom::WT controls. To improve immunohistochemical labeling of CRH neurons, mice were first treated with Colchicine. Colchicine (Sigma-Aldrich, Milwaukee, WI; 4 mg/mL in KPBS) was injected into the right lateral ventricle by using a glass micropipette. 24 hr after Colchicine treatment, mice were perfused transcardially with the same fixative as described above for GLP-1 immunohistochemistry. Brain sections were incubated for 72 hr at 4°C in blocking buffer containing the following antisera: mouse anti-HuC/D (a pan-neuronal marker to identify the cytoarchitecture and borders of the PVH; 1:500; Life Technologies, Carlsbad, CA), and either a rabbit anti-CRH (PBL#rC68; generous gift from Drs. P. Sawchenko and W. Vale, Salk Institute, La Jolla, CA), or a rabbit anti-Oxytocin antiserum (1:2500; Peninsula Laboratories, San Carlos, CA). The primary antibodies were localized with corresponding Alexa Fluor conjugated secondary antibodies (Life Technologies). Sections were mounted onto gelatin-subbed slides and coverslipped with ProLong antifade mounting medium (Life Technologies, Carlsbad, CA).