Mouse anti vglut2

Animals were sacrificed after deeply anesthetized with isoflurane, total proteins from the lumbar enlargements of spinal cords were extracted by ice-cold Radio Immunoprecipitation Assay (RIPA) lysis. Proteins were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, United States). After blocking with 5% nonfat milk, the samples were incubated with rabbit anti-β-actin (1:3000, Abcam, United States), rabbit anti-GAPDH (1:3000, Merck Millipore, United States), mouse anti-VGluT2 (1:2000, Abcam, United States), goat anti-Iba-1 (1:2000, Wako, Japan), rabbit anti-GFAP (1:2000, Abcam, United States), rabbit anti-BDNF(1:2000, Abcam, United States), or rabbit anti-TrkB (1:2000, Abcam, United States) at 4 °C overnight. The next day, the corresponding rabbit, goat, or mouse Horseradish peroxidase (HRP)-conjugated secondary IgG (1:20000, Jackson ImmunoResearch, United States) was incubated for 1 h at room temperature. The gray value of the protein on the membrane of the corresponding molecular size was detected using western blotting detection kit (WesternBright Sirius, Advansta, United States) and ChemiDoc XRS imaging system (Bio-Rad). Image Lab 3.0 system (Bio-Rad) was utilized to perform standardized analysis on the test results.

Twelve weeks after completion of cisplatin treatment, half of the mice from Experiment 2 were perfused intracardially with ice-cold PBS followed by 4% paraformaldehyde in PBS. Brains were post-fixed in 4% paraformaldehyde for 6 hours, cryoprotected in sucrose, and frozen in optimal cutting temperature compound (O.C.T.; Sakura Finetek, Torrance, CA). Sagittal sections (8 μm) were incubated with the following antibodies: rabbit anti-synaptophysin (1:1000; Millipore), mouse anti-vGLUT2 (1:1000; Abcam, Cambridge, MA), rabbit anti-PSD95 (1:1000; Abcam), mouse anti-vGAT (1:100; Abcam), and rabbit anti-gephyrin (1:1000; Abcam) followed by Alexa-488 goat anti-rabbit (1:1000; Invitrogen, Grand Island, NY) for synaptophysin, Alexa-488 goat anti-mouse (1:500; Invitrogen) for vGLUT2 and vGAT, and Alexa-594 goat anti-rabbit (1:500; Invitrogen) for PSD95 and gephyrin. As a negative control, primary antibody was omitted. Sections were visualized using a Leica SPE confocal microscope. Expression of each synaptic marker was quantified in 3–4 sections per mouse and 4–5 mice per group. The mean intensity of fluorescence was calculated using ImageJ software.

Rat primary cortical cultures were established in black-walled, glass bottom 96-well plates (μClear, Greiner Bio-One) with the same cell density and culture conditions as described for MEA experiments. Following a 4-day treatment with extract from AD patient, ALB01, or vehicle (media alone), the cultures were washed with ice cold PBS and fixed with 4% paraformaldehyde in PBS. Following fixation, the cells were permeabilized with 1% Triton X-100 in donkey serum (Jackson ImmunoResearch). The cells were probed with primary antibodies for tau (1:200, A0024, Dako), followed by anti-rabbit Cy3 secondary antibodies (Jackson ImmunoResearch) and 1 μg/mL DAPI (Thermo Fisher). Mouse anti-Vglut2 (Abcam, 1:1000) and rabbit anti-HOMER1 (Synaptic Systems, 1:1000) were used to quantify VGLUT2 and HOMER1 in aged (Day 21) iNs transduced with the lentivirus (Lenti-eGFP or Lenti-GM2A) at MOI = 5 on Day 5 or treated with TBS or AD brain extract (ALB01) from Day 17. Analysis was performed using Fiji (National Institutes of Health) by counting the number of puncta along each neuritic segment (~ 10 um), and 15 images were counted in each condition. Co-localization of puncta was defined by overlap within a circular area covering 0.52 um². Images were acquired at 40X using an LSM710 confocal microscope (Zeiss).