Anti chat

To characterize iPSC-derived neuronal cultures, neurons were fixed with 3.7% FA in 4% sucrose 14 days after the start of differentiation (d14). After blocking and permeabilization, neurons were stained with primary and secondary antibodies as described before [41 ]. The following antibodies were used: anti-TAU (rabbit, K9JA, Dako (Jena, Germany), A0024), anti-MAP2 (chicken, Abcam (Camebridge,. UK), ab5392), anti-vGlut1 (mouse, Merck Millipore (Burlington, MA, USA), MAB5502), anti-ChAT (rabbit, Thermofisher Scientific, PA5-26597), anti-SYP (mouse, Santa Cruz Biotechnology (Dallas, TX, USA), sc-17750), anti-vGlut1 (rabbit, Synaptic Systems (Göttingen, Germany), 135303), anti-PSD95 (mouse, Biolegend (San Diego, CA, USA), MMS-5182), anti-rabbit IgG Alexa-Fluor-488 (donkey, Thermofisher Scientific, A21206), anti-mouse IgG Alexa-Fluor-488 (donkey, Thermofisher Scientific, A21202), anti-chicken IgG AlexaFluor-647 (goat, Thermofisher Scientific, A21449), anti-rabbit IgG AlexaFluor-568 (goat, Thermofisher Scientific, A11011), anti-mouse IgG AlexaFluor-568 (donkey, Thermofisher Scientific, A10037).

The spinal cord was fixed in 4% paraformaldehyde and rinsed in 0.01 M PBS. Sectioned paraffin tissues were deparaffinized, and immunohistochemistry was performed as described previously [9 (link)]. The primary antibodies used were anti-CHAT (1 : 500; Thermo Scientific, Wilmington, DE, USA), anti-Iba1 (1 : 2,000; Fujifilm Wako chemical, Richmond, VA, USA), and anti-GFAP (1 : 5,000; Agilent Technologies), and the secondary antibody was an anti-horseradish peroxidase-conjugated mouse or rabbit IgG (GenDEPOT, Katy, TX, USA). The spinal cord slides were incubated with anti-mouse or rabbit IgG biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) using diaminobenzidine (Vector Laboratories). diaminobenzidine-stained slides were dehydrated with serial ethanol and xylene solutions and mounted with a coverslip and mounting solution. Images were captured using an Olympus BX51 microscope, and the intensity of the primary antibodies was quantified using ImageJ software.