Ab136253

Organoids were fixed in 4% PFA for 30 min, infiltrated with sucrose, and embedded in Optimal Cutting-Temperature (OCT) media (Fisher Scientific, Waltham, MA, USA) for cryo-sectioning. Seven-micron thick sections were taken and blocked in 1%BSA/PBS-0.3% Triton X100 for 1 h at room temperature. Organoid sections were incubated either with PSD95 (ab12093, abcam, Waltham, MA, USA), NG2 (ab275024, abcam), Olig2 (ab136253, abcam), or Abca1 (PA1 16789, Thermo Fisher) antibodies diluted 1:250 in blocking solution at 4 °C overnight. Slides were washed and incubated for three hours at room temperature in appropriate secondary antibody (A11055, Invitrogen; ab6718, abcam) diluted 1:500 in blocking solution. Slides were washed twice for 5 min in PBS, incubated for 5 min in 1:1000 DAPI, washed twice more with PBS, and cover slipped. Slides were imaged using a confocal microscope (Zeiss LSM 880 confocal microscope, Carl Zeiss AG, Oberkochen, Germany).

Lysis of cells was performed using lysis buffer (200 μL/well). Constituents of lysis buffer were NaCl (150 mM), Tris (pH 7.6, 50 mM), Triton X-100 (1%), including phosphatase and protease inhibitors. Extracted proteins (40 μg of the total proteins) were separated by performing SDS–PAGE (7.5%). Procedure of western blotting was carried out as described by Waraich et al. and Run et al. [26 (link),27 (link)]. Following antibodies were used for western blotting: anti-GFAP, Abcam (ab7260); anti-Oligo2, Abcam (ab136253); anti-Iba1, Abcam (ab5076); anti-beta actin, Abcam (ab115777); anti-caspase-3, Abcam (ab13847); anti-cleave caspase-3, Abcam (ab32042); anti-caspase-9, Abcam, (ab202068); anti-cleave caspase-9, Abcam (ab25758); anti-tau, Abcam (ab76128); anti-tau phospho, Abcam (ab109390); anti-CaMKII, Abcam (ab22609); and anti-CaMKII phospho, Abcam (ab171095). Samples of protein were transferred to nitrocellulose membrane post-electrophoresis. Membrane was blocked by bovine serum albumin or skim milk for 2 h and later incubated overnight with primary antibody (Ab) at 4°C. After washing, membrane was incubated with secondary-Ab (anti-rabbit/mouse Ig-G conjugated to horseradish peroxidase) for 1 h at room temperature. Enhanced chemiluminescence was carried out to visualize protein expression. All western blot experiments were in triplicate. ImageJ was used for protein quantification.

Following steps were performed during preparation of samples: fixation, permeabilization, labeling, and mounting. After extracting the mouse brain, it was fixed in 4% paraformaldehyde at 4°C for 24 h, followed by 30% sucrose solution storage for 24 h at room temperature. Brain tissue was further embedded in cryofreezing medium and stored at −80°C for 1 day. For microscopic study 20 μm thick sections of brain tissues were made. Following antibodies were used for immunofluorescence GFAP antibody, Abcam (ab7260, 1:800); Oligo2 antibody, Abcam (ab136253, 1:1,000); and Iba1 antibody, Abcam (ab5076, 1:750). Before proceeding to primary antibodies, sections were washed three times with phosphate buffer saline buffer for 5 min each. Sections were incubated overnight in primary antibodies at 4°C, followed by appropriate conjugated secondary antibodies for 1 h. Zeiss LSM700 was used to obtain images from the cortex region.