Fluoromount g with dapi

E98 and U87 cells, grown on Nunc Lab-Tek chamber slides (Sigma-Aldrich) to 40 % confluence, were fixed with 2 % PFA in 0.1 M phosphate buffer (pH 7.4) for 15 min at RT, followed by three washes with PBS, 30 min glycine incubation (100 mM in PBS) to quench PFA-induced autofluorescence, three PBS washes and permeabilization with digitonin for 10 min (100 μM in PBS). Aspecific binding was blocked by incubation with 20 % normal goat serum in PBS/1 %BSA for 20 min, and cells were incubated overnight at 4 °C in a humidified chamber with PBS/1 %BSA containing (combinations of): mouse monoclonal anti-CD44 antibody (as a surface marker, clone Hermes-1, ThermoFisher Scientific); rabbit anti-MET (clone D1C2, CST); rabbit anti-P-MET (clone D26, CST); mouse anti-EEA-1 (clone 14/EEA1, BD Biosciences, early endosome marker); anti-CLIMP-63 (clone G1/296, rough ER marker, a kind gift of J. Fransen). Primary antibodies were detected using Alexa Fluor 488- or 594-labeled secondary goat-anti-mouse and Alexa Fluor 488- or 568-labeled goat-anti-rabbit IgGs (all Life Technologies), 1:200 diluted in PBS/1 %BSA. Q-nuclear deep red (1:200, Life Technologies) was used to stain nuclei, and cells were mounted in Fluoromount G with DAPI (Southern Biotech, Birmingham, AL, USA). Cells were analyzed using a confocal laser scanning microscope (Leica SP2 CLSM) and Leica confocal software.

Spinal cord or brain sections mounted on slides used for immunofluorescence were rinsed in PBS for 5 min to remove residual OCT from the embedding process. After washing, samples were blocked in serum of the host of the secondary antibody (5% serum and 0.3% BSA in PBS with 0.2% Triton-X 100) and then incubated overnight with rabbit anti-mouse Iba1 (1:500, Wako), mouse anti-mouse iNOS (1:500, BD Biosciences), mouse anti-mouse Arg-1 (1:500, BD Biosciences), rabbit anti-mouse NG2 (1:500, a generous gift from the Levine lab), mouse anti-mouse CC1 (1:100, EMD Millipore), or rabbit anti-mouse GST-pi (1:250, MBL International) in 0.3% BSA in PBS with 0.2% Triton-X 100. After washing with PBS, sections were incubated with fluorescence-conjugated Alexa Fluor 488 or 555 goat anti-rabbit or goat anti-mouse antibody for 1 h at room temperature, washed three times with PBS, and mounted using Fluoromount-G with DAPI (Southern Biotech, USA). The sections were imaged at 63 × using a Zeiss LSM 510 confocal microscope. Images were acquired at the same six pre-designated locations along the ventral columns of the lumbar spinal cord section for each biological replicate.