Lsm 880 confocal imaging system

Peripheral blood CD14⁺ monocytes were plated on 24-well culture plates with coverslips. After incubation, the medium was aspirated and cells were washed twice in PBS, fixed in 4% paraformaldehyde for 15-30 minutes, permeabilized in 0.2% Triton X-100 for 10 minutes at room temperature for the exposure of intracellular antigen, and blocked in PBS containing 3% bovine serum albumin (BSA; Affymetrix) for 30 minutes. Cells were stained with a mouse anti-human CD14 monoclonal antibody, rabbit anti-human DC-STAMP, rabbit anti-human CD36, mouse anti-human clathrin, and FITC-labeled phalloidin overnight at 4°C. Alexa Fluor 594–conjugated goat anti-rabbit IgG and Alexa Fluor 488–conjugated goat anti-mouse IgG (1:1,000; concentrations of stock solutions 2 mg/ml) (life science) were added and incubated for 1 hour at 37°C. After washing in PBS, the nucleus was stained with 4’6-diamindino-2-phenylindole (DAPI, Sigma-Aldrich) and coverslips were mounted with ProLong^® Gold Antifade Reagent (Life Technologies). The images were analyzed using a Zeiss LSM 880 Confocal Imaging System (Zeiss, Jena, Germany) (29 (link)).

Organoids were collected, post-fixed in 4% paraformaldehyde (PFA, in 0.1 M phosphate buffer, pH 7.4) at 4 °C for 12 h, and then dehydrated in 30% sucrose in PBS at 4 °C. Organoids were cut into 15 µm sections mounting on freezing microtome (Leica CM 1950). For immunostaining, the organoid slices were washed in PBS, permeabilized with 0.5% Triton X-100 for 15 min and blocked in a buffer containing 3% bull serum albumin and 0.3% Triton X-100 for 1 h, and incubated with primary antibodies at 4 °C overnight. The brain slices were washed with PBS and incubated with Alexa Fluor-conjugated secondary antibodies for 1.5 h at room temperature. The sections were washed with PBS three times and mounted in adhesion anti-fade medium. For immunostaining of 2D cultures, coverslips were washed with PBS for three times followed by 4% PFA for 15 min. After washing for three times, coverslips were blocked in blocking buffer containing 3% bull serum albumin and 0.3% Triton X-100 for 1 h, and then incubated with primary antibodies at 4 °C overnight. The coverslips washed by PBS and labeled with the secondary antibodies for 1.5 h, and finally washed with PBS and then mounted in adhesion anti-fade medium. The immune-stained cells were viewed under a Zeiss LSM 880 confocal imaging system (Zeiss, Germany) at an objective magnification of 20 × . ImageJ software was used to calculate the positive cells.