Sc 33673

The total hippocampus protein was extracted by RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF) on ice and quantified using a BCA protein assay kit. Each sample (30 µg total protein) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with Tris-buffered saline containing 5% skim milk for 2 h at RT and incubated with primary antibodies at 4 °C overnight. Subsequently, the membranes were washed three times with TBST (0.1% Tween-20) and incubated with HRP-labeled secondary antibodies for 2 h at RT. The bands were detected using an enhanced chemiluminescence (ECL) system and analyzed by Image J Software. The primary antibodies used were as follows: iNOS (1:500, sc-7271, Santa Cruz, CA, USA), CD206 (1:500, sc-70586, Santa Cruz, CA, USA), GFAP (1:500, sc-33673, Santa Cruz, CA, USA), C3 (1:500, sc-28294, Santa Cruz, CA, USA), proBDNF (1:500, sc-65514, Santa Cruz, CA, USA), TrKB (1:500, sc-377218, Santa Cruz, CA, USA), BDNF (1:1000, 28205-1-AP, proteintech, Wuhan, China), p75 (1:500, sc-271708, Santa Cruz, CA, USA) and β-actin (1:1000, #4967, CST, Danvers, MA, USA). The secondary antibodies were HRP-conjugated anti-mouse (1:1000, #7076, CST, MA, USA) and HRP-conjugated rabbit (1:1000, #7074, CST, MA, USA).

Sagittal spinal cord sections were processed for immunofluorescence staining as previously described [18 (link),19 (link)]. Sections were incubated with antiglial fibrillary acidic protein (anti-GFAP) (1:100, sc-33673, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-ionized calcium binding adaptor molecule 1 (anti-Iba-1) (1:100, sc-32725, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies in a humidified chamber O/N at 37 °C. Sections were then incubated with conjugated antirabbit Alexa Fluor-488 secondary antibody #A32731 (1:1000 in PBS, vol/vol Molecular Probes, Monza, Italy) for 1 h at RT. Nuclei were stained by adding 2 μg/mL 4′,6′-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) in PBS. Sections were observed at 40× magnification using a Leica DM2000 microscope (Leica, Milan, Italy). Contrast and brightness were established by examining the most brightly labelled pixels and applying settings that allowed clear visualization of structural details while keeping the highest pixel intensities close to 250. The same settings were used for all images obtained from the other samples that had been processed in parallel.

The cultured cells and each spinal cord slides were prepared for fluorescence microscopy by permeabilization for 5 min with 0.1% Triton- × 100, blocked with 5% BSA then incubated overnight with anti-iNOS (1:100; ab49999, Abcam Cambridge, MA, USA), anti-Arg1 (1:100; ab133543, Abcam Cambridge, MA, USA), anti-C3 (1:100; 21337–1-AP, ProteinTech, Manchester, UK), anti-S100A10 (1:100; 11250–1-AP, ProteinTech, Manchester, UK), or anti-GFAP (1:100; sc-33673, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were incubated with secondary antibody, Alexa Fluor 488 goat anti-mouse IgG (H + L) antibody/Alexa Fluor 594 horse anti-rabbit secondary antibody (all 1:1000; Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. A DAPI solution was applied for 5 min for nuclear staining. Images were captured using a Leica TCA SP8 confocal laser scanning microscopy (Leica, Germany) [26 (link)].

Brain sections were processed for immunofluorescence staining as previously described [27 (link)]. Sections were incubated with the following primary antibodies: anti-GFAP (1:100; sc-33673; Santa Cruz Biotechnology), anti-Iba-1 (1:100; sc-32725; Santa Cruz Biotechnology) in a humidified chamber O/N at 37 °C. After washes with PBS solution, sections were incubated with the secondary antibody, Alexa Fluor™ (1:1000 in PBS v/v, Molecular Probes, Altrincham, UK), Invitrogen, for 1 h at 37 °C. Subsequently, brain sections were washed in PBS and nuclear staining with 4′,6′-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) (2 µg/mL) was added. Sections were observed and acquired at 40 × magnifications using a Leica DM2000 microscope (Leica, UK, EU). All stained sections were observed and analyed in a blinded manner.

Immunohistochemical staining was performed as described previously [45 (link)]. The sections were incubated overnight with a rabbit/mouse polyclonal antibody against GFAP; SC-33673 (1:300, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), IBA-1; NB100-1028, iNOS; NB300-605 (1:300; Novus Biologicals, Inc., Littleton, CO, USA), COX-2; #12282 (1:300; Cell Signaling Technology, Inc., Beverly, MA, USA). To prevent nonspecific staining, a blocking step was included. Sections were incubated at room temperature for 2 h with 5% bovine serum albumin [46 (link)] (in PBS), and incubated for overnight at 4 °C with the primary antibody in blocking solution (5% BSA). Immunohistochemical staining was performed on 8 mice per group (3 sections per each mouse).