Ab136648

ENS cultures of 5XFAD and wild type littermates were fixed with 4% paraformaldehyde (PFA) for 20 min followed by formaldehyde quenching with 50 mM NH₄Cl for 10 min. Cells were permeabilized with PBSTx (0.3% TritonX) for 15 min and blocked for 1 h at RT with FishBlock (0.1% Ovalbumin, 0.3% TritonX, 0.5% Fish gelatine in PBS). Primary antibodies for the centrosomes (Rb anti-pericentrin, Abcam ab4448, 1:500) and the ciliary membrane (Mm anti-Arl13b, Abcam ab136648; 1:200) were diluted in FishBlock and incubated overnight at 4 °C. Conjugated secondary antibodies Alexa Fluor 488 and 555 (1:400; Molecular Probes) and DAPI (Invitrogen) were incubated for 1 h at RT before mounting the coverslips on superfrost slides with Fluoromount-G^®® (SouthernBiotech, Birmingham). Immunolabelling was imaged on a Leica DM6000B fluorescent microscope (Leica, Bensheim, Germany). Deconvolution (BlindDeblur Algorithm, one iteration step) and processing of the images was performed with the Leica imaging software LAS X. Cilia length measurements were performed blinded regarding treatment or genotype using Fiji/ImageJ software (NIH, Bethesda, Rockville, MD, USA). A cilium was counted if a double staining of Arl13b and Pericentrin was visible and the length was measured via Arl13b signal in double stained cilia.

For immunofluorescence analysis, the following primary antibodies were used: mouse anti-ARL13B (1:1,000 dilution, Abcam, #Ab136648), Goat anti-IBA1 (1:500, Abcam, #Ab5076), mouse anti-IB4 (1:400, Vector Laboratories, #B-1205), rabbit anti-GFAP (1:3,000, Dako, #Z0334), rabbit anti-S100β (1:1,000, Proteintech, #15146-1-AP), rat anti-BrdU (1:1,000, Abcam, #ab6326), rabbit anti-CUX1 (1:500, Santa Cruz Biotechnology, #sc-13024), rat anti-CTIP2 (1:500, Abcam, #ab18465), rabbit anti-RSPH9 (kindly gifted from Zhu Xueliang, Shanghai Institute of Biochemistry and Cell Biology, CAS), rabbit anti-RSPH3 (1:1,000, Proteintech, #17603-1-AP).

Mice were anesthetized by using a mixture of Rompun/Zoletil and transcardially perfused with 4% paraformaldehyde in PBS. Brains were post‐fixed overnight at 4°C and slowly dehydrated by using increasing concentrations of sucrose in PBS at 4°C. Brains were embedded with Tissue‐TEK (O.C.T, Sakura Finetek), frozen in isopentane, and stored at −20°C. Sectioning was then performed with a cryostat. Antigen retrieval was applied before immunostaining, and then, sections were permeabilized using 0.3% Triton X‐100 in PBS for 30 min and quenched with 0.1 M glycine for 30 min. Sections were incubated with mouse anti‐Arl13b (ab136648, Abcam, 1:300) and/or rabbit anti‐ɣ‐tubulin (T5326, Sigma‐Aldrich, 1:1,000) primary antibodies, overnight at 4°C, and then with secondary antibodies for 1 h at RT in a solution of 0.2% gelatin, 300 mM NaCl, and 0.3% Triton X‐100 in PBS. DNA was stained in the last wash using DAPI for 30 min at RT and mounted using ProLong antifade reagent (Thermo Scientific). Imaging was performed using a Leica TCS SP5‐AOBS 5‐channel confocal system (Leica Microsystems) equipped with a 405‐nm diode, an argon ion, and a 561‐nm DPSS laser. Fixed cells were imaged using a PLAPO 40X/1.2 NA oil‐immersion objective. All the images were analyzed by using Bitplane Imaris software.