Topro3 nuclear stain

Slides from WT and GIT1 KO femure fractures were deparaffinized in xylene, rehydrated in graded ethanol, and rinsed in distilled, deionized H₂O as described above. For PECAM1 (CD31) detection, slides were boiled for 15 minutes in citrate buffer (Zymed Laboratories, San Francisco, CA), cooled for 20 minutes, and washed in PBS for 5 minutes. Slides were then blocked with normal goat serum containing 5% BSA in PBS for 30–60 minutes and incubated overnight with primary antibodies diluted 1∶100 in blocking buffer. After two rinses with PBS, the sections were incubated in the dark for 1 hour at 37°C with rabbit secondary antibody diluted 1∶500 in normal rabbit serum. Slides were rinsed in PBS before the addition of Topro3 nuclear stain (Invitrogen, Grand Island, NY) and then mounted with Prolong Gold mounting media (Invitrogen). Confocal images were captured using Zeiss LSM 510 Axioskop 2 microscope (Zeiss Microimaging, Thornwood, NY) and analyzed with Zen 2007 software (Zeiss, San Diego, CA).

HEK-293T cells were seeded at 7 × 10⁴ cells/well on 12-mm coverslips precoated with laminin/poly-d-lysine (Millicell EZ slide, Millipore) and cultured as described before (14 (link)). Cells were fixed in 4% (w/v) paraformaldehyde/PBS, blocked in 5% (v/v) FBS in PBS, and stained in blocking solution for 2 h. Primary antibodies were anti-LRRK2 (Abcam), anti-γ-tubulin (Abcam), anti-α-tubulin (Sigma-Aldrich). After three washes in PBS, the cells were incubated for 1 h with FITC- and TRITC-conjugated secondary antibodies and TO-PRO-3 nuclear stain (Invitrogen). After additional washing steps, the cells were analyzed by confocal microscopy (Zeiss LSM 710).