Anti tau1

Mice brains at the end of experiments were isolated for post fixation overnight in the 4% paraformaldehyde at 4 °C and embedded in paraffin. Five-micrometer-thick coronal sections were deparaffinized followed by epitope retrieval with citrate buffer. After treating with 3% H₂O₂, primary antibodies were incubated for overnight at 4 °C. Immunostaining was performed by liquid DAB-substrate-chromogen system (DAKO,Cat. K3467, Santa Clare, CA, USA) for 10 min and counterstain with hematoxylin (Sigma-Aldrich, Cat. GHS316, Burlington, MA, USA) for 10 s. Slides were stained with anti-Tau1 (Sigma-Aldrich, Cat. MAB3420, Burlington, MA, USA), anti-MAP2 (Abcam, Cat. ab5392), anti-synapsin (Abcam, Cat. ab64581, Cambridge, UK), and anti-ki-67 (Abcam, Cat. ab15580, Cambridge, UK) antibodies.

Cultured neurons were fixed in 4% PFA/4% sucrose, permeabilized with 0.1 M PBS containing 0.1% Triton X-100 for 10 min, and blocked for 1 hour with 1% bovine serum albumin in PBS at room temperature. Similarly, they were incubated with primary antibodies overnight at 4 °C and secondary antibodies for 1 hour at room temperature. Primary antibodies include anti-Tau1 (1:200; Millipore), anti-TuJ1 (1:500; Millipore and Abcam) and anti-MAP2 (1:500; Millipore).

cDNA encoding mouse Linx (clone 6826287, GenBank accession number BC096531) was purchased from Open Biosystems and subcloned into pcDNA3.1 (Invitrogen) and pRetroQ (Clontech) vectors to fuse Linx with the V5 and SF tag, respectively. GFP-Rho-kinase cDNA was provided by M. Amano and K. Kaibuchi (Nagoya University). The following antibodies were used in this study; anti-Linx (Islr2; R&D Systems), anti-Linx (Islr2; Abnova), anti-Ret51 (IBL, Gumma, Japan), β-actin (Sigma), anti-Tau-1 (Millipore), anti-TrkA (Cell Signaling Technology), anti-phospho-MLC (Ser19; Cell Signaling Technology), anti-MLC (Cell Signaling Technology), anti-Rho-kinase 2 (ROCK2, Abcam), anti-L1 (Millipore), anti-E-cadherin (Cell Signaling Technology), anti-Na⁺/K⁺-ATPase (Abcam), and anti-GFP (MBL, Nagoya, Japan).

Neurons were fixed in 4% PFA, then coverslips were treated for 10 min with 50 mM NH4Cl and incubated in blocking buffer (0.22% gelatin, 0.1% Triton X-100 in PBS) for 30 min, before incubation with primary antibodies for 1 h at room temperature in blocking buffer. For CB1R staining, neurons were incubated for 30 min at 37°C with anti-CB1R antibody, then rinsed three times and fixed to continue the procedure with other antibody staining. The primary antibodies used were: chicken anti-MAP2 (1:10,000, Abcam), mouse anti-ankyrinG (1:100) from NeuroMab, anti-Tau-1 from Millipore (1:1000) and rabbit anti-CB1R (1:50) from Cayman (Cat. 101500). The secondary antibodies used were a donkey anti-mouse, anti-rabbit or anti-chicken Alexa-Fluor 488, 594, or 647 (1:1000). Phalloidin Alexa-Fluor 594 was used at a concentration of 1:100. Nuclei were stained using 4′,6-diamidino-2-phenylindole, and coverslips were mounted in Fluoromount G. Images were acquired on a vertical Axioskop-2 plus microscope (Zeiss) or a Leica SP5 confocal microscope under the same conditions to compare intensities. Figures were prepared for presentation using the Adobe CS4 software.

For the collection of embryos, C57Bl/6 females were bred with Hb9 V5-PFN1^C71GWt/Tg males, using a timed mating protocol, where the time of plug check was considered to be E0.5. Pregnant females were euthanized by cervical dislocation at the appropriate timepoint. Embryos were harvested and immersion fixed in ice-cold 4% paraformaldehyde (PFA) for 90–120 min. Adult mice were anesthetized and transcardially perfused with ice-cold 1× phosphate-buffered saline (PBS). Brains were collected and fixed overnight in 4% PFA at 4°C.
The tissues were processed with an ethanol and xylene gradient in an Excelsior tissue processor (Thermo). Whole embryos were embedded in paraffin blocks. Spinal cords were cut into 2 mm segments and embedded into paraffin blocks with cervical to sacral sections arranged in a grid-like pattern. Samples were sectioned with a 5 μm thickness on a microtome (Leica). Sections were rehydrated and stained as previously described (Ittner et al., 2016 (link)). Antibodies used in this study were: anti-ChAT (Millipore), anti-profilin 1 (Abcam), anti-tau1 (Millipore), anti-TDP-43 (Proteintech) and anti-V5 (Invitrogen, Carlsbad, CA, USA).