Goat anti mouse alexa633

Cells were processed as described before26 (link) and in supplementary material using a Leica SP5 confocal microscope (Leica Microsystems). The primary and secondary antibodies used in this work were obtained from the same batches and always utilized at the same dilutions. The following antibodies were used: rabbit anti-ASMase (Santa Cruz Biotechnologies, USA) with a dilution of 1:50 overnight in humid chamber at room temperature, rat anti-LAMP-2 (Hybridoma Bank, Iowa, USA) with a dilution of 1:50 during 2 h at room temperature, rabbit polyclonal antibody anti-sortilin (kindly provided by Dr. Claus Petersen, University of Aarhus, Denmark) with a dilution of 1:50 over night in humid chamber at room temperature. Mouse anti-EEA1 (BD Bioscience) with a dilution of 1:50 over night in humid chamber at room temperature, mouse anti-GM130 (BD Bioscience) used with a dilution 1:100, 2 h at room temperature and mouse anti-Syntaxin6 (BD Bioscience) with a dilution of 1:50 over night in humid chamber at room temperature. Secondary antibodies used for indirect immunofluorescence were: goat anti-rabbit Alexa633 (dilution 1:600), goat anti-mouse Alexa633 (dilution 1:600), goat anti-rabbit Alexa488 (dilution 1:600) and goat anti-rabbit Alexa546 (dilution 1:600) (Invitrogen, USA). Secondary antibodies goat anti-Rat Cy3 and goat anti-Rat Cy5 were from Jackson Research Inc, USA.

Preparation of tissue for cryosectioning and immunofluorescence were performed as described.⁴ (link) 12 μm cryosections were obtained with a Cryostat HM560. Primary antibodies used were: chicken anti-GFP (Abcam, ab13970) at 1:2000, mouse anti-mCherry (Clontech, 632543) at 1:450, rabbit anti-dsRed (Clontech, 632496) at 1:300, mouse anti-chondroitin sulfate (Sigma, C8035) at 1:300, rabbit anti-Laminin (Sigma, L9393) at 1:200, rat anti-BrdU (Novus Bio, NB500-169) at 1:300. Secondary antibodies used were: goat anti-chicken-Alexa 488 (Thermo Fisher, A-11039), goat anti-mouse-Alexa 555 (Thermo Fisher, A-21424), goat anti-rabbit-Alexa 555 (Thermo Fisher, A-21428), goat anti-mouse-Alexa 633 (Thermo Fisher, A-21046), goat anti-rat-Alexa 633 (Thermo Fisher, A-21094) at 1:1000.

Immunofluorescence detection of LiDGATs expressed in yeast cells with myc-tag and/or FLAG-tag was carried out according to the protocol of [54 (link)]. Mouse monoclonal anti-myc antibody (Sigma Aldrich) and rabbit monoclonal anti-FLAG antibody (Sigma Aldrich) were used as primary antibodies at dilution 1:50 in 0.1 M PBS pH 7.4 with 1% (w/v) bovine serum albumin (BSA). The incubation with one (single localization experiments) or mixture of two primary antibodies (double localization experiments) was carried out overnight at 4 °C. Next day the samples were washed three times with 0.1 M PBS buffer, pH 7.4, and incubated with secondary antibodies, respectively, goat anti-mouse Alexa 633 and/or goat anti-rabbit Alexa 546 (Thermo Fisher Scientific) diluted 1:100 in the same buffer with 1% (w/v) BSA. Incubation with secondary antibodies was carried out for 2 h at RT in the dark with gentle agitation. After washing three times with the PBS buffer samples were suspended in Prolong gold anti-fade reagent (Thermo Fisher Scientific) and immediately analysed with confocal microscope.

Brains of adult female flies were dissected in PBS and immediately fixed in 4% paraformaldehyde at 4°C for 2 h as previously described (Morón-Oset et al, 2019 (link)). Brains were washed in PBS with 0.5% Triton X-100 (PBT) at RT and blocked in PBT with 5% fetal bovine serum and 0.01% sodium azide for 1 h at RT, then incubated with mouse monoclonal anti-GA (1:3,000; AB_2728663; Merck Millipore), 5H9 rat anti-polyGR (1:50 [Mori et al, 2013 (link)]) or rabbit polyclonal anti-PR (1:1,000; catalog #23979-1-AP; Proteintech) antibodies overnight at 4°C. After washes in PBT at RT, brains were incubated overnight at 4°C at 1:1,000 dilution with Alexa488 goat anti mouse (catalog #A11001; Thermo Fisher Scientific), Alexa633 goat anti-mouse (AB_2535718; ; Thermo Fisher Scientific), Alexa488 goat anti-rabbit (catalog #A11008; Thermo Fisher Scientific) or Alexa488 goat anti rat (catalog #A11006; Thermo Fisher Scientific). Finally, brains were washed in PBT, incubated in glycerol-PBS, and mounted in VectaShield antifade mounting medium with DAPI (catalog #H-1200; Vectorlabs).

Brains of adult female flies were dissected in PBS and immediately fixed in 4% paraformaldehyde at 4 °C for 2 h. Brains were washed in PBS with 0.5% Triton X-100 (PBT) at RT and blocked in PBT with 5% fetal bovine serum and 0.01% sodium azide for 1 h at RT, then incubated with mouse monoclonal anti-GA (1:3000; Merck Millipore, AB_2728663) or rabbit polyclonal anti-cleaved caspase 3 (CC3) (1:500; Cell Signaling, AB_2341188) antibody overnight at 4 °C. Following washes in PBT at RT, brains were incubated overnight at 4 °C at 1:1000 dilution with Alexa633 goat anti-mouse (ThermoFischer, AB_2535718) or Alexa488 goat anti-rabbit (ThermoFischer, catalog #A11008). Finally, brains were washed in PBT, incubated in glycerol-PBS and mounted in VectaShield antifade mounting medium with DAPI (Vectorlabs, catalog #H-1200).

Fly heads were dissected and fixed in 4% paraformaldehyde phosphate buffer saline (PBS) for 20 min at room temperature. After washing, retinas were finely dissected, permeabilized and depigmented in 0.3% (v/v) Triton X-100 in PBS (0.3% PBT) overnight at 4 °C under gentle agitation. After blocking with 5% normal goat/donkey serum in 0.3% PBT, samples were incubated overnight at 4 °C with the primary antibodies diluted in 0.3% PBT. The following antibodies were used: anti-NA/K ATPase alpha subunit (a5, DSHB, RRID:AB_2166869, 1/100), anti-rhodopsin (4C5, DSHB, RRID:AB_528451, 1/200) and anti-GFP (132004, Synaptic System, RRID:AB_11041999, 1/100). After washing, they were incubated overnight at 4 °C with Alexa 555 Phalloidin anti-F-actin (A34055, ThermoFisher Scientific) and the secondary antibodies diluted in 0.3% PBT: Alexa 488 Donkey anti-guinea pig (706-545-148, RRID:AB_2340472, Jackson ImmunoResearch), Alexa 633 Goat anti-mouse (A-21052, RRID:AB_2535719, ThermoFisher Scientific). After washing, samples were incubated in 90% glycerol PBS for 30 min in the dark before being mounted in the same solution. Retinas were imaged with a LSM710 confocal microscope (Zeiss, Wetzlar, Germany) equipped with a 40X oil objective.