Chicken anti map2

Mice were deeply anesthetized and perfused as previously described [11 (link)]. The brain was dissected, post-fixed in 4% PFA overnight, stored in PBS with 0.01% sodium azide, and sectioned at 50 μm using Leica vibratome (Leica VT1000S, Leica Inc., Nussloch, Germany). Floating sections were processed for immunocytochemistry [10 (link)]. All antibodies were obtained from Abcam (Cambridge, MA, USA) unless otherwise stated. In this study, chicken anti-GFP (1:500), rat anti-GFAP (1:1000; Invitrogen), rabbit anti-Iba1 (1:500; Wako), TMEM 119 (1:500), mouse anti-CCR2 (1:500), and rabbit anti-CD31 (1:500) were used.
For postmortem human samples, slides were baked for 60 min at 60 °C, deparaffinized with xylene for 5 min, and rehydrated in ethanol (100, 95, 70, and 50%). Antigen retrieval was performed as previously reported [17 (link)]. In this study, chicken anti-Map 2 (1:200, Abcam, Cambridge, MA, USA), rat anti-GFAP (1:1000; Invitrogen, Thermo Fisher Scientific, Rockford, IL, USA), rabbit anti-Iba1 (1:500; Wako), TMEM 119 (1:1000), mouse anti-CCR2 (1:500), and rabbit anti-CD31 (1:500) were used.

Proximity ligation assays (PLAs) were performed essentially as the manufacture instructions (Sigma-Aldrich). Briefly, neurons were fixed in 4% paraformaldehyde in PBS and probed with mouse anti-FUS (1:200, Proteintech) and anti-SNPH (1:200, Proteintech), and signals developed using a Duolink In Situ Orange kit (Sigma-Aldrich). Following PLAs, neurons were immunolabeled for chicken anti-MAP2 (1:1000, AbCam). Images were taken at × 60 (oil) on a Nikon Ti-E Two Camera microscope. Images were analysed in ImageJ and positive puncta counted using the cell counting tool. Five somas for each image were analysed for the soma count and 3 different neurites for each of five cells per image were analysed with three biological replicates carried out.

The primary antibodies included the following: rabbit anti-Ki67 (#15580, Abcam), mouse anti-beta III tubulin (TUJ1, #1637, Millipore), mouse anti-GFAP(#N206A/8, NeuroMab), mouse anti-NEUN (#MAB377, Millipore), chicken anti-MAP2 (#5392, Abcam), rabbit anti-PH3 (#9701, Cell Signaling), rabbit anti-TBR2 (#23345, Abcam), rabbit anti-cleaved caspase-3 (#9661, Cell Signaling), and rabbit anti-PAX6 (#PRB-278P, Covance). The secondary fluorochrome-conjugated antibodies were diluted 1:400 (donkey anti-mouse, donkey anti-rabbit, goat anti-mouse and goat anti-rabbit, goat anti-chicken (Invitrogen]). Nuclear counterstaining was performed with 4’, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, #B2261at 0.25 μg/μl). TUNEL staining was carried out using a kit purchased from Roche (#12156792910). Images were obtained using an LSM710 confocal microscope (Zeiss).

Motor neurons, matured for 50 days, and transiently transfected HEK293T cells at 72-h post transfection, were used for immunocytochemistry. Cells were washed and fixed with 4% paraformaldehyde and blocked for 1 h in Phosphate-buffered saline (PBS) containing 0.2% Triton X-100 and 10% normal donkey serum for iPSC-MNs and 5% normal goat serum for HEK293Ts respectively (Jackson ImmunoResearch). Samples were then incubated overnight at 4 °C with primary antibodies. The next day, cells were washed with PBS with 0.1% Triton X-100. Samples were then incubated with the appropriate secondary antibodies (diluted in blocking solution) conjugated to Alexa488, Alexa555 or Alexa647 fluorophores (1:500 to 1:1000 Molecular Probes) for 1 h at RT. Cell nuclei were labeled by DNA staining using DAPI or Hoechst (Life Technologies).
Primary antibodies Chicken anti-MAP2 (1:5000, Abcam ab5392); Goat anti-ChAT (1:500); Rabbit anti-MATR3 (1:200; Abcam 151714); Mouse Anti-digoxin (1:200; Jackson Immunoresearch 200-002-156); Rabbit anti-FLAG (1:500, Sigma F7425); Rabbit anti-Histone H3 (1:200; Abcam ab1791)

Sections were washed three times with 1× PBS and then incubated with primary antibodies at 4 °C overnight, after 1 h blocking by 5% normal goat serum (NGS, Sigma, Darmstadt, Germany) and 0.2% Triton X-100 (Sigma, Darmstadt, Germany). The sections were then incubated at room temperature for 2 h with fluorescence conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) and washed with 0.01 M PBS (1×) 3 times before being observed under a confocal laser scanning microscope (Zeiss, LSM800, Jena, Germany). Fluorescence immunohistochemistry was performed by using the following primary antibodies: mouse anti-NF160 (Abcam, 1:400, Cambridge, UK), rabbit anti-NeuN (Abcam, 1:500, Cambridge, UK), chicken anti-Map2 (Abcam, 1:500, Cambridge, UK), rabbit anti-Gfap (Dako, 1:1000, Carpinteria, CA, USA), rabbit anti-Sox2 (Abcam, 1:200, Cambridge, UK), chicken anti-Gfap (Abcam, 1:1000, Cambridge, UK), guinea-pig anti-Iba1 (Synaptic Systems, 1:800, Göttingen, Germany), mouse anti-Tuj1 (R&D, 1:400, Minneapolis, MN, USA), and mouse anti Nestin (Abcam, 1:1000, Cambridge, UK).