Mouse anti nos2

DRG tissues for assessing neuroprotective effect of minoxidil on paclitaxel-induced neuropathy were isolated at Day1, 4, or 7 after first paclitaxel injections. Sequentially, DRG tissues were fixed with 4% PFA for 4 hours and dehydrated with 30% sucrose in PBS overnight at 4 °C. For frozen sections, the samples were embedded in OCT (Leica, IL, USA) and sliced into 20 μm thick sections. These DRG tissue sections were washed with 0.05% Triton X-100 in PBS for 30 minutes and blocked with 3% BSA at room temperature for 1 hour. DRG tissue samples were co-stained with mouse anti-NeuN antibody (1:100; Millipore), mouse anti-NOS2 (1:100; Santa Cruz), mouse anti-Iba-1 (1:100; Santa Cruz), rabbit anti-Arginase-1 (1:100; Santa Cruz), rabbit anti-Iba-1 (1:100; Abcam) and rat anti-CD68 antibody (1:100; Novus, USA) at 4 °C overnight. Then, these samples were washed by PBS and incubated in secondary antibodies (Alexa-488 and Alexa-594 1:200; Invitrogen, CA) at room temperature for 1 hour. Immunofluorescence images of DRG tissues were acquired using a 40x objective by Olympus FV1000 confocal microscope (Olympus, Japan).

For immunofluorescence analysis, cells were plated on coverslips, exposed to treatments and processed as previously described [40 (link),41 (link),42 (link),43 (link),44 (link)]. After washing with PBS, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, Milan, Italy) for 20 min at room temperature. After fixation, cells were washed three times in PBS for 5 min and blocked in Odyssey blocking buffer for 1 h at room temperature. Subsequently, cells were incubated with mouse anti-arginase 1 (Cat. No. sc-271430, Santa Cruz Biotechnology, Heidelberg, Germany) and mouse anti-NOS2 (Cat. No. sc-7271, Santa Cruz Biotechnology), overnight at 4 °C. The following day, cells were washed three times in PBS for 5 min and incubated with the following secondary antibodies: anti-mouse TRITC-conjugated secondary antibody (Cat. No. A11003, ThermoFisher Scientific, Milan, Italy), and anti-mouse FITC-conjugated secondary antibody (Cat. No. F2012, Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. Antibodies were diluted in Odyssey blocking buffer. Slides were mounted with mounting medium containing 4,6-diamidino-2-phenylindole (DAPI, Cat. No. D1306, Invitrogen, 1:1000) to highlight nuclei. Fluorescent images were obtained using a Zeiss Axio Imager Z1 microscope with an Apotome 2 system (Zeiss, Milan, Italy).