Rabbit polyclonal anti iba1

Human and murine RPE and retinal flatmounts and human and murine sections were stained and quantified as previously described (Sennlaub et al, 2013 (link)) using polyclonal goat anti-human APOE (Millipore), polyclonal rabbit anti-IBA-1 (Wako), polyclonal rabbit anti-rat FASL (Millipore), monoclonal rat anti-mouse IL-6 (R&D Systems), AlexaFluor 555 phalloidin (Mol probes), and rat anti-mouse CD102 (clone 3C4, BD Biosciences Pharmingen) appropriate secondary antibodies and counterstained with Hoechst if indicated. Preparations were observed with fluorescence microscope (DM5500, Leica) or a FV1000 (Olympus) confocal microscope. Histology of mice eyes and photoreceptor quantification were performed as previously described (Sennlaub et al, 2013 (link)).

Mice were deeply anaesthetized by i.p. injection of pentobarbital and perfused transcardially with PBS, followed by ice-cold 4% paraformaldehyde/PBS. The L3–4 (transverse) segments of the spinal cord were removed, postfixed in the same fixative for 3 h at 4 °C, and placed in 30% sucrose solution for 24 h at 4 °C. Transverse L4 spinal cord sections (30 μm) were incubated in blocking solution (3% normal goat serum or normal donkey serum) for 2 h at room temperature and then incubated for 48 h at 4 °C with primary antibodies: polyclonal rabbit anti-PAX2 (1:1000, Invitrogen), monoclonal rat anti-GFAP (1:2000, Invitrogen), monoclonal mouse anti-NeuN (1:2000, Millipore), polyclonal rabbit anti-Iba1 (1:5000, Wako), and polyclonal rabbit anti-c-Fos (1:5000, Santa Cruz). Following incubation, tissue sections were washed and incubated for 3 h at room temperature in secondary antibody solution (Alexa Fluor 488 and/or 405, 1:1000, Molecular Probes). The tissue sections were washed, slide-mounted and subsequently coverslipped with Vectashield hardmount (Vector Laboratories). Three to five sections from the L4 spinal cord segments of each mouse were randomly selected and analysed using a LSM 700 Imaging System (Carl Zeiss).

This methodology was carried out in accordance with a previously established protocol [34 (link)]. To begin, mice were transcardially perfused with a fixative containing 4% paraformaldehyde, and their brains were subsequently cryoprotected with 30% sucrose. Following this, sagittal free-floating brain sections, each 60 μm in thickness, were incubated overnight at 4 °C with polyclonal rabbit anti-Iba1 (Wako Chemicals, Cat.#: 019-19741) at a 1:1000 dilution. Subsequently, the sections were incubated for 90 min with Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Cat.#: A11011) at a 1:500 dilution. This was followed by a 15 min incubation in DAPI at a 1:1000 dilution, and a 14 min wash in PBS. The immunofluorescence labeling was then detected using a confocal microscope.