Cy3 conjugated anti mouse

Immunostaining was performed as described in Durand-de Cuttoli et al., 2018 (link), with the following primary antibodies: anti-tyrosine hydroxylase 1:500 (anti-TH, Sigma, T1299) and chicken anti-eGFP 1:500 (Aveslab, AB_10000240). Briefly, serial 60 μm-thick sections of the midbrain were cut with a vibratome. Slices were permeabilized for one hour in a solution of phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA, Sigma; A4503). Sections were incubated with primary antibodies in a solution of 1.5% BSA and 0.2% Triton X-100 overnight at 4 °C, washed with PBS and then incubated with the secondary antibodies for 1 hr. The secondary antibodies were Cy3-conjugated anti-mouse (1:500 dilution) and alexa488-conjugated anti-chicken (1:1000 dilution; Jackson ImmunoResearch, 715-165-150 and 703-545-155, respectively). For the juxtacellular immunostaining, the recorded neurons were identified with the addition of AMCA-conjugated streptavidin (1:200 dilution) in the solution (Jackson ImmunoResearch). Slices were mounted using Prolong Gold Antifade Reagent (Invitrogen, P36930). Microscopy was carried out either with a confocal microscope (Leica) or with an epifluorescence microscope (Leica), and images were captured using a camera and analyzed with ImageJ.

Primary neuron cultures were generated following procedures described previously [21 (link), 22 (link), 27 (link)]. Briefly, embryos were dechorionated using bleach, selected at approximately stage 11, sterilized with ethanol and mechanically dissociated. Cells were then chemically dispersed, washed in Schneider’s medium with 20% fetal calf serum and plated onto concanavalin A (5 μg/ml) coated glass coverslips. Coverslips were kept on custom incubation chambers, where cells were grown as hanging-drop cultures at 26 °C for 3–10 days in vitro (DIV). Primary neurons were fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS; pH 6.8 or 7.2) for 30 min at room temperature and then washed three times in PBS with 0.3% Triton X-100 (PBT), followed by staining. Antibody staining and washes were performed in PBT using anti-tubulin (clone DM1A, mouse, Sigma-Aldrich, 1:1000, RRID:AB_477583) and anti–GFP (ab290, rabbit, abcam, 1:1000, RRID:AB_303395). Secondary antibodies were anti-rabbit Alexa Fluor 488 (1:1000, RRID:AB_2576217, goat) and Cy3-conjugated anti-mouse (1:200; Jackson Immuno-Research, RRID:AB_2315777, donkey). Culture slides were mounted in ProLong Gold.

Minimum essential medium (α-MEM) and Dulbecco modified minimal essential medium (D-MEM) was obtained from Gibco Laboratories (Buenos Aires, Argentina). Fetal bovine serum (FBS) was purchased from Natocor S.A. (Córdoda, Argentina). The inhibitors α-difluoromethylornithine (DFMO) and Chloroquine (CQ) and the TRITC-conjugated phalloidin was purchased from Sigma (Buenos Aires, Argentina). The rabbit anti-Beclin-1 antibody was purchased from Santa Cruz (Santa Cruz Biotechnology, INC) and the polyclonal rabbit anti-LC3 antibody from Sigma (Buenos Aires, Argentina). The following antibodies were also used: anti-Atg5 (Abcam), anti-p62 (Abcam), Anti-ubiquitin (Santa Cruz), anti-β-actin (Genescript) and the anti-β tubulin (E7) was obtained from Developmental Studies Hybridoma Bank. The secondary antibodies Cy3-conjugated anti-mouse and Cy3-conjugated anti-rabbit were purchased from Jackson Immuno Research Laboratories, INC, as well as the antibodies HRP-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG and HRP-conjugated Anti-goat IgG. Hybond-ECL nitrocellulose membranes were from Amersham. The DNA marker Hoechst 33342 was purchased from Life Technologies.

HEK-293T cells were fixed in 4% paraformaldehyde for 15 min and then washed twice with PBS containing 20 mM glycine before permeabilization with the same buffer containing 0.2% Triton X-100 (5 min incubation). The cells were treated for 1 h with PBS containing 1% bovine serum albumin and labeled with a mouse anti-Rluc (1/100; MAB4400, Millipore) antibody and subsequently treated with Cy3-conjugated anti-mouse (1/200; 715-166-150; Jackson ImmunoResearch (red)) IgG secondary antibody (1 h each). Alternatively, cells were labeled with rabbit anti-D₃R (#ab42114), anti-MT₁R (#ab203038), or anti-MT₂R (#ab115336) antibodies, all from Abcam (Cambridge, UK) and subsequently treated with Cy3-conjugated anti-rabbit (1/200; #711-166-152; Jackson ImmunoResearch (red)) IgG secondary antibody (1 h each). The samples were washed several times and mounted with 30% Mowiol (Calbiochem). Samples were observed under a Leica SP2 confocal microscope (Leica Microsystems).

HEK-293T cells were seeded on glass coverslips in 12-well plates. Twenty-four hours later, cells were transfected with CB₂R-YFP cDNA (1 μg) and/or OX₁R-Rluc cDNA (1 μg). Forty-eight hours after, cells were fixed in 4% paraformaldehyde for 15 min and washed twice with PBS containing 20 mM glycine before permeabilization with PBS-glycine containing 0.2% Triton X-100 (5 min incubation). Cells were blocked during 1 h with PBS containing 1% bovine serum albumin. HEK-293T cells were labeled with a mouse anti-Rluc antibody (1/100, MAB4400, Millipore, Merck, Darmstadt, Germany) and subsequently treated with a Cy3-conjugated anti-mouse (1/200, 715-166-150 (red), Jackson ImmunoResearch, St. Thomas Place, UK) IgG secondary antibody (1 h each). The CB₂R-YFP expression was detected by the YFP’s own fluorescence. Nuclei were stained with Hoechst (1/100 from stock 1 mg/mL; Sigma-Aldrich). Samples were washed several times and mounted with 30% Mowiol (Calbiochem). Images were obtained in a Zeiss LSM 880 confocal microscope (ZEISS, Germany) with the 63X oil objective.