Goat anti guinea pig alexa 488

The primary and conjugated antibodies used in this study are listed in Table S1. Secondary antibodies used for Western blot were goat anti-mouse IgG HRP (BioRad; 1:3000) and polyclonal goat anti-Rabbit IgG HRP (Dako; 1:5000). Secondary antibodies used for immunofluorescence were donkey anti-rabbit IgG Alexa 594 (Invitrogen A21207; 1:400), goat anti-mouse IgG Alexa 568 (Invitrogen A11004; 1:200) and goat anti-guinea pig Alexa 488 (Invitrogen A11073; 1:200). The PP2 and PP3 compounds were purchased from Merck Chemical Ltd. EGF and PDGF-BB were obtained from Sigma-Aldrich. HGF was obtained from R&D Systems and staurosporine was from Tocris Bioscience. The polyclonal rabbit antibodies against the phosphorylated Y1440 and Y1422 sites on β4 are homemade (method described in supplemental materials).

About 10⁶ differentiated cells were dissociated into single-cell with 0.25% trypsin or accutase at 37 ^oC. Intracellular antibody staining was performed using Becton Dickinson Cytofix/Cytoperm and Becton Dickinson Perm/Wash buffer according to manufacturer instructions. The following concentrations of primary and secondary antibodies were used: anti-FOXA2 (rabbit IgG, 1:500, GeneTex); anti-SOX17 (mouse IgG, 1:500, R&D Systems); anti-PDX1 (goat IgG, 1:500, R&D Systems); anti-insulin (guinea-pig IgG, 1:500, Dako); donkey anti-rabbit-Alexa 568,1:1000 (Invitrogen, Carlsbad, CA, USA), donkey anti-mouse-Alexa 488, 1:1000 (Invitrogen), donkey anti-goat-Alexa 488,1:1000 (Invitrogen) and goat anti-guinea pig-Alexa 488,1:1000 (Invitrogen). The cells were then washed with FACS buffer (PBS contains 2% fetal bovine serum, FBS). Control samples were stained with isotype-matched control antibodies. The cells were washed and resuspended in FACS buffer and then processed for analysis on FACS Accuri C6 (BD) or FlowJo software.

The following antibodies and concentrations were used: guinea pig anti-perilipin 1:1500 (Fitzgerald 20R-PP004); rabbit anti-Mac2 1:500 (Cedarlane, Clone M3/38); chicken anti-GFP 1:700 (Abcam, ab13970); donkey anti-rabbit Alexa 647 1:200 (Invitrogen, A-31632); goat anti-guinea pig Alexa 488 1:200 (Thermo Fisher Scientific, A-11073); goat anti-chicken Alexa 488 1:200 (Invitrogen, A-11039); and goat anti-guinea pig Alexa 647 1:200 (Invitrogen, A-21450). Paraffin sections were dewaxed and hydrated in xylene and 100–95–80–70–50% ethanol and ddH₂O. Slides were placed in chambers containing 1× R-Buffer A pH 6.0 solution and antigen retrieval was performed using Antigen Retriever 2100 (Electron Microscopy Sciences) for 2 h. Following one PBS wash for 5 min, Fx Signal Enhancer (Invitrogen) was added to the slides for 30 min at room temperature. Slides were then blocked for 30 min in PBS containing 10% normal goat serum at room temperature. Primary antibodies were diluted in PBS containing 10% normal goat serum and added to paraffin sections overnight at 4 °C. Following overnight incubation, slides were washed in PBS and incubated with secondary antibodies diluted in PBS containing 10% normal goat serum for 2 h at room temperature. Washed slides were mounted with Prolong Anti-Fade mounting medium containing DAPI (Invitrogen) before images were acquired for analysis.

The following antibodies were used for immunofluorescence (IF) and/or immunoblotting (IB) at the indicated dilutions: Rabbit anti-peIF2α (for PLA 1:6000 #44728G Invitrogen, and IB, 1:1000, Cell Signaling), mouse anti-eIF2α (IB, 1:1000, Cell Signaling). peIF4B, p4EBP1, peIF4G (IB:1:2500, cell signaling). Most of the commercially available antibodies that should recognize rodent HRI failed to robustly detect the protein in neurons. Nevertheless, the use of HRI knock-out tissue allowed us to identify in neurons, after immunoprecipitation, an HRI-specific band only with one HRI antibody: 07–728, Millipore. Rabbit anti-actin (1:5000, Abcam), rabbit anti-H3 (1:10000, Abcam) rabbit anti-biotin (IB, 1:1000), mouse anti-puromycin (IB, 1:1000, IF 1:3500, Kerafast,), guinea pig anti-MAP2 (IF 1:1000, Synaptic Systems), rabbit anti- HA-tag (IF 1:2000, Rockland) Goat anti-mouse or anti-rabbit IR680 or IR800 (IB, 1:5.000, Licor), goat anti-guinea pig Dylight405 (IF 1:1000, Dianova), goat anti-guinea pig-Alexa488, and goat anti mouse-Alexa546 or -Alexa488, goat anti-rabbit Alexa647 or -Alexa546 (IF all 1:1000, ThermoFisher).

Fly heads were cut open, fixed in 2% formaldehyde, and washed with 0.5% PAXD buffer (1X PBS, 5% BSA, 0.03% sodium deoxycholate, 0.03% Triton X-100). The fixed heads were dissected, and the isolated brains were permeabilized in 1% PBT for 20 min and then blocked in 0.5% PAXD containing 5% horse serum for 1 h. The following primary antibodies were added to the mixtures directly: anti-DTk antibody (Gp2) (Lee et al. 2021 ); anti-NC82 antibody (DSHB), diluted 1:200. The brains were washed with PAXD and incubated overnight with secondary antibodies in a blocking solution at 4°C. The following secondary antibodies were used at a 1:200 dilution: goat anti-guinea pig Alexa-488 (Thermo Fisher Scientific), goat anti-mouse Alexa-555 (Thermo Fisher Scientific). Stained brain samples were washed with PAXD, incubated in 0.1 M phosphate buffer containing 50% glycerol for 30 min, and mounted using a mounting medium. Confocal images were obtained using an LSM 800 confocal microscope (Carl Zeiss) and were processed using Zen software (ZEN Digital Imaging for Light Microscopy, Carl Zeiss). DTk intensity was determined using the ImageJ software. In the dorsal fan-shaped body (dFB), DTk fluorescence signal above the background was selected by adjusting the color threshold. The total intensity of DTk in the dFB was obtained by multiplying mean intensity and the area of the selected region.

The following antibodies were used for immunofluorescence (IF) and immunoblotting (IB) at the indicated dilutions: rabbit anti-biotin (IB 1:1000, Sigma), guinea pig anti-MAP2 (IF 1:1000, Synaptic Systems), anti-rabbit IRDye680 (IB 1:5000, Licor), goat anti-guinea pig Dylight405 (IF 1:500-1:1000, Jackson ImmunoResearch), and goat anti-guinea pig-Alexa488 (IF 1:1000, Thermo-fisher).