Paraformaldehyde pfa p6148

We obtained lovastatin (PHR1285), mevalonate (41288), 25-hydroxycholesterol (H1015), MG-132 (M8699), Filipin (F9765), geranylgeraniol (G3278), protease inhibitor cocktail (P8340), N-Ethylmaleimide (E3876), and paraformaldehyde (PFA) (P6148) from Sigma; [¹⁴C]-acetic acid sodium salt (NEC084H001) from Perkin Elmer; and FuGENE HD transfection reagent (E2312) from Promega; G418 (345810), digitonin (300410), henylmethylsulfonyl fluoride (PMSF) (52332), leupeptin (108975), pepstatin A (516481) and ALLN (208719) from Merck; Hoechst 33342 (H1399) from Invitrogen. Lipoprotein-deficient serum (LPDS) [41 (link)] and delipidated-fetal calf serum (FCS) [42 (link)] was prepared from FCS (S1580, Biowest) by ultracentrifugation in our laboratory [25 (link)].

Compound libraries of ion channel ligands were obtained from Enzo drug bank (BML-2805; ENZO Life Sciences, NY, USA). Primary mouse DRG neurons were incubated with compounds at final concentration of 1 μM for 24 hours, and then with 0.01 or 0.1 μM paclitaxel for another 24 hours. After paclitaxel treatment, primary DRG cells for neurite outgrowth analysis were washed by PBS and then fixed with 4% paraformaldehyde (PFA, P6148; Sigma-Aldrich) for 15 mins. These fixed DRG cells were washed with 0.05% Triton X-100 in PBS for 30 minutes and blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich, USA) at room temperature for 1 hour. Subsequently, DRG cells were stained with goat anti-β III tubulin (SC-9935, 1:100; Santa Cruz, USA) and mouse anti-NeuN monoclonal antibody (1:100; Millipore, USA) overnight at 4 °C. The samples were then washed by PBS and reacted with secondary antibodies (Alexa-488 and Alexa-594 1:200; Invitrogen, CA) at room temperature for 1 hour. For image acquisition and analysis of DRG neurite outgrowth, images of stained cells were automatically acquired using 20X objective by ImageXpress^Micro wide field fluorescent microscope (Molecular Devices, USA) as previously described11 (link).