Donkey anti mouse 647

Cultured cells were fixed by immersion in 4% (w/v) PFA in PBS overnight at 4 °C and PBS washed before primary antibodies were applied in blocking solution (10% FCS/0.2% Triton X-100 in PBS) overnight at 4 °C. After PBS washing, Alexa Fluor-conjugated secondary antibodies, diluted in blocking solution, were applied overnight at 4 °C. Coverslips were washed thrice and mounted with DAKO fluorescent mounting medium (Sigma). Primary antibodies included: rabbit anti-Pcdh15^{32 (link)} [0.45 mg/ml; 1:200; Figs. 1 and 2], rabbit-anti-pan Pcdh15 (HL5614^{29 (link)}, 1:100; Fig. 8), rabbit anti-CD3 Pcdh15 (PB375^{29 (link)}, 1:100; Fig. 8), rat anti-GFP (Nacalai Tesque, 1:2000), goat-anti-PDGFRa (R&D System; 1:100), mouse-anti-Arp2 (Santa Cruz Biotechnology SC376698; 1:10) and mouse-anti-Arp3 (Santa Cruz Biotechnology SC48344; 1:10). Secondary antibodies included: donkey-anti-rat 488 (Invitrogen; 1:1000), donkey-anti-rabbit 568 (Invitrogen; 1:1000), donkey-anti-rabbit 647 (Invitrogen; 1:1000), donkey-anti-rabbit 488 (Invitrogen; 1:1000), donkey-anti-mouse 647 (Invitrogen; 1:1000) and donkey anti‐goat 647 (Invitrogen; 1:1000). Filamentous (F)-actin was also detected by exposing cells to Alexafluor-568 conjugated phalloidin (Invitrogen; 1:50) and cell nuclei were detected by exposing cells to Hoechst 33342 (Thermo Fisher Scientific; 1:1000).

Slides were warmed to room temperature for 20 min and then given three washes in 1× PBS for 10 min each. After which slides were incubated for 1 h with 10% Protein Block, serum-free (Dako) in 1× PBS. Slides were then incubated overnight at room temperature with a primary antibody in a solution of 1% Protein Block, 1% bovine serum albumin, and 99.9% 1× PBS with 0.1% Triton X-100. Primary antibodies were as follows: mouse anti-chondroitin-4-sulfate (C4S; 1:400; Millipore), Wisteria floribunda agglutinin (WFA; 1:1000; Vector Labs), mouse anti-parvalbumin (1:1000; Swant), rabbit anti-parvalbumin (1:1000; Swant); rabbit anti-IBA1 (1:200; Dako); mouse anti-GFAP (1:200; Sigma-Aldrich); c-Fos (1:400; Cell Signaling); mouse anti-GAD67 (1:400; Millipore); anti-gephyrin (1:500; ThermoFisher). After overnight incubation, slides were washed three times, twice in 1× PBS with 1% Tween 20 and once in 1× PBS. Slides were then incubated for 1 h with secondary antibodies in antibody solution (as above). Secondary antibodies were as follows: streptavidin 647 (1:200; Invitrogen), donkey anti-mouse Alexa Fluor 488 (1:200; Invitrogen), donkey anti-rabbit Alexa Fluor 647 (1:200; Invitrogen), and donkey anti-mouse 647 (1:200; Invitrogen). After 1 h of incubation, slides were washed again three times and mounted with 4’,6-diamidino-2-phenylindole (DAPI) in Vectashield mounting medium (Vector Labs).

Free-floating sections were blocked for 2 h at RT in 3% BSA and 0.3% Triton in Phosphate-buffered saline (PBS) and incubated with primary antibodies [rabbit anti-TMEM119; 1:100 (Abcam), mouse anti-CD11c; 1:100 (Abcam), rat anti-CD169; 1:200 (BioLegend) rabbit anti-CD3; 1:100 (Abcam), mouse anti-CD45; 1:500 (BD biosciences), mouse anti-CD68; 1:500 (Abcam), mouse anti-CD317; 1:100 (R&D Systems), mouse anti-CS-56; 1:200 (Abcam), rat anti-GFAP; 1:200 (Thermo Fisher Scientific)], mouse anti-NeuN; 1:100 (Millipore), for 48 h at 4°C. Sections were washed for 3 × 30 min in PBS and incubated for 2 h at RT with secondary antibodies [Donkey anti-mouse 568, 1:500 (Invitrogen); Donkey anti-rat 488, 1:500 (Invitrogen); Donkey anti-rabbit 568, 1:500 (Invitrogen)]; Donkey anti-mouse 647, 1:1,000 (Invitrogen) together with DAPI (1:1,000). After a second round of washing (3 × 30 min in PBS), sections were mounted with Fluorogold prolong antifade mounting medium (Invitrogen). The list of material used has been summarized in Supplementary Table 2.

After dissection, Drosophila midguts were fixed in 4% paraformaldehyde in 1X PBS for 45 to 90 minutes and successively washed 3 times with 0.1% TritonX in PBS. Guts to be immunostained were then incubated for an hour in blocking solution (1% bovine serum albumin, 1% normal donkey serum, and 0.1% Triton X-100 in PBS). Overnight primary antibody staining was performed at RT. Guts were washed 3 times with 0.1% TritonX in PBS and ≥2 hour secondary antibody staining was performed in PBS. Primary antibodies used: rabbit anti-pH3 (1:000, EMD Millipore), rabbit anti-β-Galactosidase (1:1000, MP Biomedicals), and mouse anti-Prospero (1:100, DSHB). Secondary antibodies used: donkey anti-rabbit-555 (1:2000, Thermo Fisher), donkey anti-mouse-488 (1:2000, Thermo Fisher), and donkey anti-mouse-647 (1:1000, Thermo Fisher). DNA was stained in 1:50,000 DAPI (Sigma-Aldrich) in PBS and 0.1% TritonX for 30min, and samples received a final three washes in PBS before mounting in antifade medium (Citifluor AF1). Imaging was performed on a Zeiss LSM 700 fluorescent/confocal inverted microscope.

Brain was harvested as above and coronal sections of 60 μm were prepared with a freezing microtome (Leica) and stored in a cryoprotectant solution (25% glycerol, 30% ethylene glycol, in PBS). Before staining, slices were washed in PBS. Sections were blocked in 5% normal donkey serum (Jackson ImmunoResearch) in PBST for 60 min at room temperature followed by incubation with primary antibody: mouse anti-HA (1:500; ThermoFisher, A26183) overnight at 4 °C. Sections were subsequently washed three times in PBST (5 min each) and then transferred to a secondary antibody solution, donkey anti-Mouse 647 (1:500; ThermoFisher, A31571) for 3 h at room temperature. Samples were stained with DAPI, washed three times with PBST for 5 min each, and then imaged. Confocal fluorescence imaging was performed on an Olympus FV3000 confocal microscope.