Alexa 594 conjugated secondary antibody

Cells cultured on slides were washed three times with PBS and fixed with 4% paraformaldehyde at 4°C for 30 min. After another wash, cells were permeabilized in PBS with 0.1% Triton X-100 for 15 min at room temperature and then blocked with 10% donkey serum albumin (Jackson Immuno Research Laboratories, PA, USA). Then, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with Alexa 594-conjugated secondary antibody (Jackson Immuno Research Laboratories) for 1 h at room temperature and another incubation with DAPI for 10 min. Slides were mounted with an anti-fluorescence quenching agent and observed under an Olympus fluorescence microscope.

Microglial morphology and proliferation were examined, as before (Ferreira et al., 2014 (link); Lam and Schlichter, 2015 (link); Lam et al., 2017 (link)). For imaging, cells were seeded at 8 × 10⁴ cells/coverslip, stimulated with TGFβ1 for 24 h, and fixed in 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA, USA; Cat# 15710) for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, washed, and labeled overnight with CD11b (1:2500, Bio-Rad/AbD Serotec). The next day, cells were washed in PBS and incubated for 1 h with Alexa 594-conjugated secondary antibody (Jackson ImmunoResearch Labs; Cat# 715-585-151, RRID:AB_2340855; 1:200). Cells were counterstained for 1 h with Acti-stain 488 phalloidin (1:50; Cytoskeleton Inc., RRID:SCR_013532; Cat# PHDG1-A) to visualize filamentous (F-) actin, and with DAPI (1:3000, 10 min) to label nuclei. Coverslips were mounted on glass slides using Dako mounting medium and stored in the dark at 4°C until images were acquired with a confocal microscope (model LSM880; Zeiss) and processed in ImageJ. Unipolar cells (those with a lamellum and trailing uropod) were counted using ImageJ in three micrographs each (20× magnification) and expressed as a percentage of total cells counted (~120–200/culture).