Rat monoclonal anti cd11b

Mice were deeply anaesthetised by i.p. injection of pentobarbital and perfused transcardially with phosphate buffered saline (PBS), followed by ice-cold 4% (w/v) paraformaldehyde/PBS. The L4 segments of the spinal cord, or the L4 DRG were removed, postfixed in the same fixative for 3 h at 4 °C, and placed in 30% (w/v) sucrose solution for 24 h at 4 °C. Transverse L4 spinal cord sections (30 μm) and L4 DRG sections (15 μm) were incubated in blocking solution [3% (v/v) normal goat serum] for 2 h at room temperature and then incubated for 48 h at 4 °C with primary antibodies: rabbit polyclonal anti-Iba1 (1:5000, Wako), and rat monoclonal anti-CD11b (1:1000, Serotec), rat monoclonal anti-CD31 (1:200, BD Pharmingen) and hamster monoclonal anti-CD3 (1:100, eBioscience). Following incubation, tissue sections were washed and incubated for 3 h at room temperature in secondary antibody solution (Alexa Fluor 546 and Alexa Fluor 405, 1:1000, Molecular Probes, OR, USA). The tissue sections were washed, slide-mounted and subsequently coverslipped with Vectashield hardmount (Vector Laboratories). Three to five sections from the L4 spinal cord and DRG of each mouse were randomly selected and analysed using an LSM700 Imaging System (Carl Zeiss).

The cells were fixed with 4 % PFA, and unspecific binding was prevented by incubating cells in a 3 % BSA and 0.5 % Triton X-100 solution (all from Sigma-Aldrich) for 30 min at RT. The cells were incubated with primary antibodies prepared in 0.3 % BSA and 0.1 % Triton X-100, kept overnight at 4 °C, washed with PBS, and finally incubated for 1 h at RT with the corresponding secondary antibody. Antibodies used were as follows: rat monoclonal anti-CD11b (1:600; AbD Serotec), mouse monoclonal anti-acetylated α-tubulin (1:100; Sigma-Aldrich), goat polyclonal anti-Nox1 (1:250; Santa Cruz Biotechnology, Heidelberg, Germany), Alexa Fluor 594 goat anti-rat, Alexa Fluor 488 goat anti-rat, Alexa Fluor 488 donkey anti-goat, and Alexa Fluor 594 rabbit anti-mouse (all 1:200 in PBS, from Life Technologies Ltd). Membrane ruffling was observed using phalloidin, a marker for filamentous actin. The cells were incubated for 2 h with Alexa Fluor 594 conjugated to phalloidin (1:100; Life Technologies Ltd) in PBS, at RT. For nuclear labeling, cell preparations were stained with Hoechst 33342 (2 μg/ml, Life Technologies Ltd) in PBS, for 5 min at RT and mounted in Dako fluorescent medium. Fluorescent images were acquired using an Axio Observer LSM710 confocal microscope (Zeiss) under a 63× oil objective.