Lsm 710 inverted microscope

All staining of astrocytes for markers of endocytosis were performed in the same manner. Cells were plated in 96-well glass bottom plates (IBIDI) at a density of 10,000 cells per well. First cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature and then permeabilized using 0.2% TritonX-100 for 10 minutes at room temperature, blocked for 1 hour at room temperature using 1% normal goat serum, 1% normal donkey serum in 5% PBS. Primary antibodies were diluted according to supplier’s suggestions in blocking solution and incubated overnight at 4°C. The next day, cells were washed and incubated with secondary antibodies at 1:1000 dilution for an hour at room temperature and imaged using a Zeiss LSM 710 inverted microscope. Antibodies used were as follows: EEA1 (mouse BD biosciences 610457, 1:100), Rab5 (rabbit, CST, 1:400), Rab7 (rabbit, CST, 1:100), Rab11 (rabbit, CST, 1:50). Imaging and quantification criteria are described below.

Live red blood cell confocal imaging, before and after freezing with cryoprotectants, was undertaken using a Zeiss LSM 710 inverted microscope, equipped with three photo-multiplier detectors (GaAsP, multialkali, and BiG.2) and a multi-channel spectral imaging detector. Red blood cells (diluted 500-fold) were placed on MatTek glass bottom dishes (no. 1.5, 35 mm) and brightfield images were taken using a C-Apochromat 63x/1.20 W Korr M27 objective lens and ×3.0 zoom. Z-stacks were taken every z = 0.3 μm until the whole cell was imaged. Zeiss ZEN (black edition) 2.3 lite was utilized for image collection and maximum intensity projection processing.

RBL cells were incubated with GO in buffered salt solution (BSS: 135 mM NaCl, 5.0 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 5.6 mM glucose, and 20 mM HEPES, pH 7.4). Cell fixation was carried out with 4% paraformaldehyde in PBS for 10 minutes followed by multiple washes with 1 mg/ml BSA in phosphate-buffered saline (PBS). For F-actin labeling, fixed cells were incubated with 5 µg/mL A647-phalloidin in PBS (10 mg/mL BSA) using 0.1% v/v Triton-X100 for 20 minutes at room temperature before washing with PBS. For FN immunofluorescence, fixed cells were labeled with primary antibody (5–10 µg/mL) at room temperature for 1 h in PBS with 10 mg/mL BSA. After additional washing, fluorescent secondary antibody (5–10 µg/mL) in PBS with 10 mg/mL BSA was added to the samples at room temperature for another 1 h. A final series of PBS washes were performed before storing samples in 2 mL of PBS with 0.01% sodium azide at 4°C until ready for imaging on the microscope. Unless otherwise indicated, samples were imaged on a Zeiss LSM 710 inverted microscope with a motorized stage using a 63× Oil Plan-Apochromat objective lens. A DF 488/561/647 filter set was used to perform sequential 1/2/3 color imaging of the samples. The area of the focal plane was adjusted for optimal image quality.