Mouse anti iba1

Double-labelling were performed for Iba-1 and cleaved caspase-8, Iba1 and CD16/32 and CD16/32 and cleaved caspase-8. Two different Iba1 antibodies were used: mouse anti-Iba-1 (Millipore) to co-localize with cleaved caspase-8 and rabbit anti-Iba-1 (Wako) to co-localize with CD16/32. Sections were blocked with PBS containing 1% appropriate serums (Vector Laboratories) for 1 hour. The slides were washed three times in PBS, then incubated overnight at 4°C with the two primary antibodies to be tested diluted in PBS containing 1% appropriate serums and 0.25% Triton X-100. Sections were incubated with the following secondary antibodies: a) mouse anti-Iba1: horse anti-mouse conjugated to Texas Red (1:200; Vector Laboratories), b) rabbit anti-Iba1 and rabbit anti-cleaved caspase-8: horse anti-rabbit conjugated to Fluorescein (1:200; Vector Laboratories), c) rat anti-CD16/32: chicken anti-rat conjugated to AlexaFluor 594 (1:200, Invitrogen). Incubation with secondary antibodies were for 2 hours at room temperature in the dark. Their addition was preceded by three 10-minute rinses in PBS. Nuclei were counterstained with Hoechst dye (1 μg/ml; Molecular Probes/Invitrogen). Fluorescence images were acquired using a Zeiss LSM 7 DUO confocal laser scanning microscope (Carl Zeiss Microscopy, Jena, Germany) and processed using the associated software package (ZEN 2010; Carl Zeiss Microscopy).

The following primary reagents were used: chicken anti-PV (1:500, Synaptic Systems, Göttingen, Germany, 195006), biotinylated Wisteria floribunda agglutinin (WFA, 1:1000, Vector laboratories B-1355, Burlingame, CA, USA), rabbit anti-Bcan [32 (link),33 (link)] (1:1000), guinea pig anti-vGluT1 (1:1500, Synaptic Systems, 135304), mouse anti-Pcan (1:500, The Developmental Studies Hybridoma Bank, Iowa City, IA, USA, 3F8-c), mouse anti-Vcan (1:500, The Developmental Studies Hybridoma Bank, 12C5-c), rabbit anti-vGAT (1:500, Synaptic Systems), mouse anti-Iba1 (1:1000, Millipore, Boston, MA, USA, MABN92) and rabbit anti-Iba1 (1:1000, Wako, Richmond, VA, USA, 019-19741).
As secondary antibodies, we used Alexa Fluor 405 goat anti-chicken IgG (1:500, Abcam, Cambridge, UK, ab175675), Alexa Fluor 647 goat anti-chicken IgG (1:1000, Invitrogen, Waltham, MA, USA, A-21449), streptavidin Alexa Fluor 405 conjugated (1:1000, Invitrogen, S32351), Alexa Fluor 647 goat anti-rabbit IgG (1:1000, Invitrogen, S32351), Alexa Fluor 405 donkey anti-guinea pig IgG (1:500, Sigma Aldrich, St. Louis, MO, USA, SAB4600230), Alexa Fluor 647 goat anti-mouse IgG (1:1000, Invitrogen, A21236), Alexa Fluor 405 donkey anti-rabbit IgG (1:500, Abcam, ab175651), Alexa Fluor 488 goat anti-rabbit IgG (1:200, Abcam, ab150077), and Alexa Fluor 546 goat anti-guinea pig IgG (1:200, Invitrogen, A11074).

Equal amounts of mice spinal cord homogenates protein (40 μg) were resolved separately for soluble and membrane fractions on SDS-PAGE, transferred to nitrocellulose membrane, and blocked overnight with 5 % skim milk in TBS-T (0.3 % Tween 20). Blots of the soluble fraction were probed with the following primary antibodies: mouse anti-actin (1:10,000 Sigma-Aldrich, USA), rabbit anti-MCP-1 (1:1000 Peprotech, USA), rabbit anti-IL-6 (1:1000 Peprotech, USA), and mouse anti-Iba-1 (1:1000 Millipore, Germany). Blots of the membrane fraction were probed with the following primary antibodies: mouse anti-actin (1:10,000 Sigma-Aldrich, USA), rabbit anti-occludin (1:1000 Abcam, UK), rabbit anti-claudin 5 (1:500, Sigma- Aldrich, USA), rabbit anti-ZO-1 (1:1000 Sigma-Aldrich, USA), and rabbit anti-cd36 (1:1000 Abcam, UK). Blots were incubated with corresponding secondary antibodies conjugated peroxidase (Sigma- Aldrich, USA) and developed with the EZ-ECL detection kit (Biological Industries, Israel). Quantitative densitometric analysis was performed using the densitometric software EZQuant-Gel (version 2.12).

Free-floating sections were first washed in 0.1 M PB (3 × 15 min) at room temperature (RT) and then overnight with gentle agitation in a cold room. On the next day, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide (H₂O₂); non-specific binding blocked with 10% normal goat serum (NGS) in Tris buffered saline + Triton (TBS-T) for 30 min, and sections were incubated on a shaker table at RT with the primary antibody mouse anti-Iba1 (Millipore) at 1:1000 in 5% NGS-TBS-T for about 21 h 30 min, with the secondary antibody biotinylated anti-mouse immunoglobulin G (IgG) (Vector) made in goat at 1:500 in TBS-T for 2 h and streptavidin–horseradish peroxidase (S-HRP) conjugate (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 1:1000 in TBS-T for 2 h. Sections were rinsed in TBS-T (3 × 5 min) before and after all blocking and antibody incubation steps. Afterward, the sections were carefully developed with filtered nickel-intensified 3,3′-diaminobenzidine (DAB) (Sigma, St. Louis, MI, USA) for approximately 3 min with any excess DAB rinsed off with PB (3 × 4 min). The specimens were left in the cold room overnight; mounted the next day, and dried overnight in 37 °C. Finally, they were cleared in xylene (2 × 5 min) and coverslips were mounted using Depex.