A21430

Following viral transduction, slices were fixed overnight at 4°C in 0.1 M phosphate-buffer containing 4% formaldehyde. Then, slices were rinsed with phosphate-buffer saline (PBS), permeabilized with PBS/gelatin 0.2%/Triton 0.25%, and incubated overnight at 4°C with rabbit anti-Satb2 (1:1000, ab34735, Abcam; Lee et al., 2010 (link)) and chicken anti-GFP (1:1000, GFP-1020, Aves Labs; Tricoire et al., 2010 (link)). After washing in PBS, the respective immunoreactions were visualized with the following secondary antibodies: goat-anti-rabbit AlexaFluor 555 (1:1000, A-21430, Thermo Fisher Scientific) and goat-anti chicken AlexaFluor 488 (1:1000, A-11039, Thermo Fisher Scientific) incubated 1 h at room temperature. Sections were mounted with fluoromount-G (Southern Biotech) on slides for visualization. Images of immunostained material were acquired using a Leica TCS SP5 AOBS inverted confocal microscope with a 40× objective (40× HCX P APO CS NA 1.25–0.75/Oil) and LAS AF software (Leica Microsystems). Cell counting was performed using Image Pro Analyzer 7.0.0.951 (MediaCybernetics).

To identify the cells positive for TGF-β and TGF-β receptors, costaining was performed using a pan-leucocyte marker (CD45). Cryosections (10 µm) were fixed for 15 min in 4% PFA and endogenous peroxidases were inhibited with 0.3% H₂O₂. They were then processed as described in Supplementary Table S1. Briefly, after a 1 h blocking step, slides were incubated with primary antibodies, overnight (O/N) at 4 °C. Slides were further incubated with the ImmPRESS™ Horseradish Peroxidase reagent kit (MP7401, Vector Laboratories), and an A555-labelled tyramide (B40955, ThermoFisher Scientific) according to the manufacturer’s instructions or an A555 goat anti-rabbit antibody (2 μg/ml, A21430, ThermoFisher Scientific) for the TGF-β1 staining only. For the co-localisation with CD45, sections were further saturated for 1 h and incubated with the anti-CD45 primary antibody (550539, BD Biosciences) O/N at 4 °C before incubation with an A488-goat anti-rat IgG (2 μg/ml, A11006, ThermoFisher Scientific). Finally, slides were counterstained with 1 μg/ml Hoechst 33342, mounted in Citifluor™ Tris-MWL 4-88 solution and observed with a Leica SPE confocal microscope. Digital images were processed with the OMERO open-source software. All the antibodies shown were tested on the same lot of animals.

Immunochemical experiments were performed as previously described [25 (link)]. Briefly, the transduced HEK293T cells, iPSC lines, and neurons were fixed in 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries) for 30 min, permeabilized using 0.1% Triton X‐100 (Fujifilm Wako Pure Chemical Industries) in phosphate‐buffered saline (PBS) for 10 min, and blocked with 4% Block Ace (DS Pharma Biomedical, Osaka, Japan) in 0.01% Triton X‐100 for 1 h at room temperature (approximately 25 °C). The following primary antibodies were used: mouse anti‐microtubule‐associated protein (MAP2; 1 : 1000; ab11267; Abcam, Cambridge, UK), rabbit anti‐MAP2 (1 : 1000; ab32454; Abcam), anti‐GLP1R (1 : 50; PA5‐97789; Thermo Fisher Scientific), anti‐NPY2R (1 : 100; SAB4502029; Sigma‐Aldrich), anti‐CCKAR (1 : 100; ab115287; Abcam), anti‐GLP2R (1 : 50; MAB4285; R&D Systems, Minneapolis, MN, USA), anti‐HTR3A (1 : 250; MA5‐31771; Thermo Fisher Scientific), and Hoechst 33342 (H342; Dojindo Laboratories, Kumamoto, Japan). The following secondary antibodies were used: Alexa Fluor 555 (F[ab])2 goat anti‐rabbit IgG [H + L] secondary antibody (A21430; Thermo Fisher Scientific) and Alexa Fluor 488 (F[ab′]2 goat anti‐mouse IgG [H + L] secondary antibody) (A11017; Thermo Fisher Scientific).