Mab360

Immunohistochemistry (IHC) was performed as previously reported [51 (link)]. For immunofluorescence in cultured astrocytes, cells were fixed with 4% paraformaldehyde (15 min, 0°C), rinsed with glycine solution (30 mM in 0.1 M PBS solution), and permeabilized with Triton X-100 (0.2%, 3 min). Preincubation was performed with 10% NGS for 1 h at 37°C. The following primary antibodies were used: mouse anti-NeuN (1:1000; Abcam #ab104224), mouse anti-GFAP (1:500, Millipore #MAB360), rabbit anti-GFAP (1:500, Proteintech #16825-1-AP), guinea pig anti-S100β (1:500, Synaptic Systems #287 004), rabbit anti-Ezrin (1:100, Cell Signaling #3145), mouse anti-vGluT1 (1:200, Millipore #MAB5502), and rabbit anti-PSD95 (1:100, Invitrogen #51–6900). The following Alexa conjugated secondary antibodies were used: goat anti-mouse 488 (1:1,000, Abcam #ab150113), goat anti-rabbit-488 (1:1,000, Abcam #ab150077), goat anti-rabbit 647 (1:500, Abcam #ab150079), and goat anti-guinea pig 488 (1:500, Abcam #ab150185). Immunofluorescence of Ezrin was performed by using an Alexa Fluor 488 Tyramide SuperBoost Kit (Invitrogen #B40912 and #B40941) according to the manufacturer’s protocols. After nucleus labeling with DAPI, the coverslips were mounted on slides using anti-fade solution. For quantification of fluorescent intensity, sections from the two groups were stained and imaged with exactly the same protocol.

Protein expression in the cortex and cerebellum was quantified by western blotting. Total protein was extracted using a total protein extraction kit (Applygen Technologies Inc., P1250). Fifty micrograms of protein from each sample were separated on 10% or 12% SDS–PAGE gels and transferred onto PVDF membranes. The membranes were incubated overnight at 4°C with primary antibodies, including β-actin (1:2000, Proteintech, 60008-1-Ig), Tbk1 (1:1000, abcam, ab40676), phospho-Tbk1 antibody (1:1000, Cell signaling, 5483), p62 (1:1000, Sigma, P0067), phospho-p62 (Phospho-Ser403) (1:1000, SAB, 12804), LC3B (1:500, Sigma, L7543), TDP-43 (1:1000, Proteintech, 10782-2-AP), GFAP (1:1000, Millipore, MAB360), ubiquitin (1:1000, Proteintech, 10201-2-AP), or hSOD1 (1:10000, Abcam, ab51254). Following incubation with fluorescent secondary antibodies for 1 h at room temperature, an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) was used to detect the bands of interest.