Rabbit anti iba1 019 19741

For mouse brain staining, brains were dissected and fixed in 4% paraformaldehyde for 1 hr on ice followed by immersion in 20% sucrose overnight at 4°C. Brains were then sectioned using a freezing stage Microtome (Microm KS 34, Thermo Scientific) and spread out on glass coverslips for immunostaining. The antibodies were used at the following concentrations: mouse anti-AnkG (N106/36, N106/20) (UC Davis/NIH), 1:200; rabbit anti-ATF3 (Santa Cruz) (RRID:AB_2058590), 1:500; chicken anti-GFP (ab13970, abcam) (RRID:AB_300798), 1:1000; rabbit anti-Iba1 (019–19741, Wako), 1:250; chicken anti-GFAP (ab4674, abcam) (RRID:AB_304558), 1:500; NeuroTrace (Thermo Scientific), 1:200; Alexa 488-, Cy3-, or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch), 1:250. Samples were then mounted in Vectashield (Vector Laboratories) before being analyzed under a confocal microscope. The images were obtained with an Axio-imager Z1 microscope (Carl Zeiss) fitted with an AxioCam digital camera (Carl Zeiss). The images were analyzed by ZEN 2012 (Carl Zeiss).

For immunofluorescence analysis, mice were transcardially perfused with 4% paraformaldehyde in PBS (pH 7.4). Perfused brains were dissected, post-fixed, and cryoprotected in 30% sucrose in PBS. The brains were sectioned to obtain 30 µm-thick coronal sections that were stored in maintenance solution (30% sucrose, 0.1% NaN₂ in PBS) at 4 °C until used. Selected brain sections from the midbrain region were blocked for 30 min at RT in 10% NGS, 0.3% Triton X-100 in PBS, followed by an o/n incubation at 4 °C with 1:500 rabbit anti-IBA1 (019-19741, Wako, Richmond VA, USA) in 5% NGS, 0.3% Triton X-100 in PBS. Sections were then incubated with 1:500 anti-rabbit secondary antibody conjugated to Alexa Fluor-488 fluorophore (A11008, Thermo Fisher Scientific, MA, USA) diluted in 5% NGS in PBS for 3 h at RT. Images were acquired using a 40× objective of Leica TCS SP confocal laser-scanning microscope (Leica, Germany). Images elaboration and cell density analysis were obtained using ImageJ (NIH).

The second and third series of sections were used for immunohistochemical staining to visualize neurons and microglia. They were treated in 1% hydrogen peroxide (in 0.1 M PBS, 30 min) to quench the endogenous peroxidase. After three rinses with 0.01 M PBS, they were incubated in 1% normal goat serum for 3 h to block the non-specific binding sites. Sections were then incubated in primary antibody solution (rabbit anti-Iba1, 019-19741 from Wako Chemical, 1:500; mouse anti-NeuN, MAB377 from Merck, 1:1000) overnight at room temperature. After three rinses with 0.01 M PBS, they were incubated in the biotinylated secondary antibody solution (goat anti-mouse IgG for NeuN, 1:200, #62-6540, Thermo Fisher; goat anti-rabbit IgG for Iba1, 1:200, #65-6140, Thermo Fisher) for 2 h at room temperature. Sections were then rinsed and incubated in HRP conjugated streptavidin (1:1,000, #189733, Merck) for 2 h. The peroxidase reaction was carried out using a developer solution containing 0.4 mg/mL DAB and 0.0006% hydrogen peroxide dissolved in 0.1 M TBS. For the negative control, the primary antibodies were omitted, and all other steps carried out as described above. After the staining procedures, sections were mounted onto gelatin-coated slides and dehydrated and coverslipped with DPX mounting medium.