Conjugated secondary antibody

For analysis of regrowth defects, appropriately staged pupae were dissected out of the pupal case at 24–96 h APF and v’ada in segments A2—A4 was visualized in live animals using a Zeiss LSM710 confocal microscope. All images were analyzed using Fiji [44 (link)], the NeuronJ plugin [45 (link)] was used for dendrite length measurements. NLS-tdtomato^PEST/CD8::GFP intensity ratios of 5’ UTR reporters were measured in the soma of third instar lateroventral (v’ada) c4da neurons. lifeact::GFP/tdtomato and pHluorin/tdtomato ratios were measured in primary dendrites at the indicated timepoints. Airy scan images of GFP::RalA were taken on a Zeiss LSM880 confocal microscope, and fluorescence profiles were generated in Fiji. Adult female abdominal bodywalls were dissected dorsally in PBS, fixed in 4% formaldehyde/PBS for 20 minutes, and blocked in goat serum. Filets were incubated overnight with rabbit anti-Ppk26 antibodies [46 (link)] and chicken anti-GFP (Aves labs cat. # GFP-1020). Mical stainings of early pupae were done as described [18 (link)]. Conjugated secondary antibodies were from Invitrogen.

Cells were fixed in ice-cold 4% (mass/vol) paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS, incubated with blocking buffer consisting of 10% (vol/vol) goat serum in PBS, incubated with the primary antibodies rabbit anti-ONECUT3 (Biorbyt, orb312423, 1:100) and mouse anti-TUBB3 (Covance, MMS-435P, 1:1000) in 5% (vol/vol) goat serum in PBS at 4°C overnight, incubated with the conjugated secondary antibodies (Invitrogen) in 5% (vol/vol) goat serum in PBS at RT for 1 h, incubated with 0.01% (vol/vol) Hoechst (Invitrogen) in PBS at RT for 10 min, and finally mounted onto slides with fluorescence mounting medium (Dako). Imaging was performed using an Axio Imager Z1 (Zeiss). CellProfiler (21 ) was used to quantify staining intensity for individual cells at the nucleus.

Cells were directly grown on glass coverslips (0.17 mm thickness, 1.5 H high performance; Marienfeld Superior) placed in 24-well plates. Cells were fixed with 4% paraformaldehyde (20 min; RT), permeabilized with methanol (10 min RT) and blocked with 5% bovine serum albumin (5% BSA diluted in phosphate-buffered saline [PBS]-Triton 0.1%). Primary antibodies of interest were incubated overnight at 4°C. Secondary antibodies conjugated to Alexa fluorophores were incubated 1 hr RT in the dark. The following conjugated secondary antibodies (Invitrogen) were used: goat anti-rabbit IgG H+L (Alexa-488 or -647); donkey anti-mouse IgG H+L (Alexa-555, -561, -633, or -647). After incubation, samples were washed with PBS-T (x3) and nuclei were stained with Hoechst (25 µg/ml diluted in 5% BSA-PBS-T; 1 hr RT in the dark). Five PBS-T washes and a final MiliQ water were performed. Coverslips were mounted using Pro-long glass (Invitrogen). Preparations were maintained 24–48 hr in the dark at RT and then stored at 4°C up to image acquisition.

For immunostaining of ES cells or ECs derived from embryoid bodies, cells were fixed for 10 minutes in PBS containing PFA4% and Sucrose 4%. After a brief rinse in PBS, cells were permeabilized in 0.1% Triton for 10 minutes and then immersed in a blocking solution (10% Fetal bovine serum in PBS). Primary antibodies (see Key Resources Table in STAR Methods) were diluted in blocking solution and incubated for 2 hours at room temperature or overnight, followed by three washes in PBS of 10 minutes each and incubation for 1 to 2 hours with conjugated secondary antibodies (Invitrogen or Biotium) at room temperature. After three washes in PBS, cells were mounted with Fluoromount-G (SouthernBiotech).
For immunostaining of mouse retinas, eyes from mouse pups were dissected and fixed for 1 hour in a solution of PFA4% in PBS. After washing the tissue in PBS twice, retinas were microdissected and processed for immunostaining following a very similar protocol previously described above for the cells. The only difference is that the blocking/permeabilization buffer contains 0.3% Triton, 3% FBS, 3% Donkey Serum and antibody washes were more extended in time; on average for 30 minutes each.

For confocal analyses, we dissected at least 10 animals. Fixation and preparation of tissues for immunohistochemistry was performed as described in ref. ^{71 (link)}. Staining of larval brains was repeated at least three times (n > 10 each time). The following other antibodies were used: Anti-Repo Mab8D12 (1:5, Developmental Studies Hybridoma Bank (DSHB); anti-Repo (rabbit, 1:1000, gift of Dr. B. Altenhein, Cologne); anti-V5 (1:500, Invitrogen, R96025); anti-HA (1:1000; Covance, MMS-101P 901503); anti-HRP-DyLight^TM649 (1:500; Dianova, 123-165-021); anti-GFP (1:1000; Invitrogen, A6455); conjugated secondary antibodies (all 1:1000, Invitrogen), and phalloidin (1:100, Invitrogen, A22287). All specimens were analyzed using a Zeiss 710 or 880 LSM; orthogonal sections were taken using the Zeiss LSM imaging software.

Cells were grown on coverslips and fixed with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 in phosphate-buffered saline (PBS). Primary antibodies were incubated for 1 h at room temperature and visualized by conjugated secondary antibodies (invitrogen). Coverslips were mounted in SlowFade-Anti-Fade (Invitrogen) and examined under a confocal microscope (Leica TCS SP2)

hESC-RPE cells grown on GFR-MG or VXF were fixed with 4% paraformaldehyde/phosphate-buffered saline (1x PBS) (Sigma-Aldrich) for 10 min at room temperature, then permeabilized with 0.1% TritonX-100 (Roche, Indianapolis, IN, USA) in PBS for 3 min. Samples were then incubated with primary antibody (1:40–1:1000 dilution) in blocking buffer composed of 3% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS for 90 min at room temperature. Unconjugated primary antibodies were washed and incubated with conjugated secondary antibodies (1:500; Invitrogen) for 30 min in the dark at room temperature. Cell nuclei were co-stained using Hoechst 33342 (1:1000; Life Technologies). F-actin was stained with Alexa-Fluor-488-conjugated Phalloidin (Phalloidin-488) (1:40; Life Technologies) for 30 min in the dark at room temperature. Images were captured using a Nikon C1 Confocal microscope (Nikon Instruments Inc., Melville, NY, USA). Primary and secondary antibodies used in this study are listed in Supplemental Materials.