Lsm700 confocal microscopy

Annexin V staining was performed according to the manufacturer's instructions. In short, unstimulated, TNFα-stimulated or TNFα-stimulated in presence of FasL, adherent and non-adherent RPTEC were washed in cold phosphate-buffered saline (PBS). Washed non-adherent cells were centrifuged, the supernatant discarded, and the cell-pellet was resuspended in annexin-binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4). Adherent and non-adherent RPTEC were then diluted to a cell density of about ~1 × 10⁶ cells/ml. In situations where cell death was marginal, all non-adherent RPTEC were pelleted and subjected to analyses. Five microliters of Alexa Fluor® 488 annexin V were added to each 100 μl of cell suspension before the cells were incubated at room temperature for 15 min. Next, the cells were subjected to DAPI for 1 min and washed with annexin-binding buffer. The non-adherent cells were placed on a slide. Both the non-adherent and adherent annexin V and DAPI stained RPTEC were subjected to a Carl Zeiss LSM 700 confocal microscopy for fluorescence detection.

Longitudinal spinal cord sections were processed for immunohistochemistry as previously described (Mukhamedshina et al., 2019 (link)). Primary and fluorescence secondary Abs are described in Table 1. Nuclear stain via 4′,6-diamidino-2-phenylindole (DAPI) (10 μg/mL, Sigma). Coverslips were mounted on slides using ImmunoHistoMount medium (ab104131, Abcam). Sections incubated only with secondary Abs (without primary Abs) were used as a reaction control. The slides were examined and photographed using LSM 700 confocal microscopy (Carl Zeiss). Three-dimensional reconstruction and cell quantification/semi-quantitative analyses were performed with Zen 2012 software (Carl Zeiss).

Indirect immunofluorescence assay was performed on Hep2 antinuclear antigen tissue slides (Bion Enterprises, Des Plaines, IL). The sera were diluted at 1 : 80 in PBS and pH 7.4 and incubated with the slides for 30 min at room temperature. After extensive washing, the slides were incubated with fluorescein isothiocyanate- (FITC-) conjugated goat anti-human IgG secondary antibody (Santa Cruz Biotechnology, CA) or anti-rabbit IgG Fab2 (Alexa Fluor 488) as secondary antibody diluted 1 : 100 in PBS for 1 h at room temperature. The slides were washed two times with PBS before adding a drop of mounting media containing 1.5 μg/mL 4′,6′-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA) to prevent photobleaching. The slides were then examined under fluorescence microscopy, LSM 700 Confocal Microscopy (Zeiss), at 400x magnification. Zen 2009 software was used for images capture and analysis.

3 ×10⁵ cells were seeded on coverslip overnight. Cells were incubated at 4 °C for 1 h to depolymerize microtubules. To re-polymerize microtubules, cells were incubated in humidified atmosphere with 5% CO₂ at 37 °C for 10 min. Cells were then washed with ice-cold PBS and fixed by 100% ice-cold methanol. Cells were blocked with 3% BSA in PBST (PBS with 0.1% Trion X-100) for 30 min and incubated with primary antibody at room temperature for 2 h. Next, cells were washed with PBST for 3 times followed by incubation with secondary antibody for 1 h. Nuclei were stained with UltraCruz and images were acquired by using Model Zeiss LSM700 Confocal Microscopy with a 100×/1.40 oil objective lens (UPlanSApo).

Sections or coverslips with cells were washed with Tris buffered saline (TBS) and incubated in blocking buffer (1% glycine, 0.4% Triton X-100, 3% BSA, 0.1% sodium azide and 10% normal goat serum in TBS) for one hour at room temperature. Sections or coverslips were incubated in the primary antibodies in species-appropriate combinations for 24 hours at 4 °C. Primary antibodies and dilution used in our experiments included rabbit anti-GFP (Catalog: A-11122, 1:1500, Invitrogen), rat anti-GFP (Catalog: GF090R, 1:2000, Nacalai), mouse anti-MAP2 (Catalog: ab11267, 1:200, Abcam), Goat anti-EphB2 (Catalog: AF467, 1:250, R&D systems), rabbit anti-Ephrin-A5 (Catalog: ab70114, 1:250, abcam) and rabbit anti-RFP (Catalog: ab62341, 1:250, Abcam). After incubation with primary antibodies, sections or coverslips were washed with TBS and incubated with secondary antibodies in blocking buffer at room temperature for two hours. Secondary antibodies included Alexa 488, 546, 633 conjugated goat anti-mouse, anti-rat, anti-rabbit IgG (1:500; Abcam). Sections or coverslips were then washed with TBS and mounted with ProLong Gold anti-fade (Invitrogen). All images were taken by Zeiss LSM700 confocal microscopy. The volume, migratory distance, and the average fluorescent intensity were estimated by using ImageJ (U. S. National Institutes of Health).