Rabbit iba1

Membranes were transferred into 6 well plates and slices were fixed for 20mins in 4 % paraformaldehyde in 0.1 M PBS (applied both above and below the membrane insert). To reduce the volumes required for subsequent steps, the membranes were cut free of the plastic inserts and the sections of membrane containing the slices were transferred, using forceps, to individual wells in a 24 well plate. Slices were washed twice in TBS, blocked for 1 h in blocking solution (TBS with 0.5 % Triton X-100 and 3 % Goat Serum) then incubated in 200 μl primary antibody diluted in blocking solution overnight at 4 °C with shaking. Slices were washed 3 times in TBS before being incubated (2 hs, RT in the dark) with Alexa488, 568 or 647 conjugated secondary antibodies (Life Technologies-diluted 1:250 in blocking solution). After a final 3 TBS washes, some slices were counterstained with Thioflavin S, BTA-1, Nissl or Hoechst. Images were captured using a Nikon Confocal Microscope. Primary antibodies used: mouse Tuj1 (Covance 1:1000), rabbit Tuj1 (Sigma 1:500), chicken Tuj1 (Abcam:1:1000) rabbit NFL (Millipore 1:250), mouse MOAB2 (pan specific to Aβ- Millipore 1:1000), rabbit GFAP (Abcam 1:1000), mouse synaptophysin (Dako 1:1000), rabbit PSD95 (Abcam 1:500), rabbit tau (Dako 1:1000), rabbit Iba1 (Wako 1:500) rabbit calbindin and rabbit parvalbumin (Kind gifts from Dr P Emson 1:1000).

Fixed hemispheres of the mouse brains were cut into 35‐μm sections (coronal sections) using a Leica VT1000S vibratome. Free‐floating sections were washed with PBS three times for 5 min, blocked with 3% bovine serum albumin for 30 min, and finally incubated with primary antibodies diluted in PBS [(rabbit Iba1, 1:1,000, #016‐20001; Wako Chemicals), (rat CD68, 1:150, MCA1957; Bio‐rad)] overnight at 4°C. Incubated slices were then washed with PBS three times for 5 min, incubated for 2 hr at room temperature with a secondary antibody (1:400; Jackson Laboratories) in PBS, and then washed with PBS three times for 5 min at room temperature. Cells were stained with DAPI and mounted using fluorescence mounting medium (#S3023; Dako). Fluorescent images were taken with a Zeiss Axio Observer Z1 microscope and processed using AxioVision 4.8.2.

Cells were subjected to a 5 min wash with 1× phosphate buffered saline (PBS) and fixed for 20 min with PBS containing 4% (w/v) paraformaldehyde and 4% (w/v) sucrose, pH 7.4 at room temperature. Incubation for 5 min with PBS containing 0.1% (v/v) Triton X-100 was done to permeabilize cells. Microglia were subsequently blocked for 1 h with PBS containing 10% (v/v) goat serum and incubated overnight at 4°C with rabbit Iba-1 (1:750; Wako) in blocking buffer containing 10% goat serum. After incubation with Alexa Fluor 546 goat IgG secondary antibody (1:1000) in PBS containing 0.1% (v/v) triton X-100 and 1% goat serum, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 13.0 ng/μL) in PBS, followed by two 5 min PBS washes (Daniele et al., 2015 (link); Sánchez and Maguire-Zeiss, 2020 (link)). Coverslips were subsequently mounted with Hydromount (Electron Microscopy Services) in preparation for imaging with the Nikon Eclipse Ti-S inverted fluorescent microscope.

After treatment was stopped, cells were prepared for immunocytochemistry by washing with phosphate buffered saline (PBS) for 5 min, followed by a 20-min fixation with PBS containing 4% (w/v) paraformaldehyde and 4% (w/v) sucrose, pH 7.4 at room temperature. Cells were then permeabilized in PBS containing 0.1% (v/v) Triton X-100 for 5 min, and blocked for 1 h with PBS containing 10% (v/v) goat serum followed by overnight incubation at 4°C with rabbit anti-NFκB (1:1000; α-p65; Abcam), rabbit Iba-1 (1:750; Wako), rat CD11b (1:1000; EBT), rabbit MMP-13 (Abcam; 1:100), or rat CD68 (1:400; Bio-Rad Laboratories) in blocking buffer containing 10% goat serum. Antibody:antigen complexes were visualized following incubation with Alexa Fluor 594 or 488 conjugated goat IgG secondary antibody (1:1000) in PBS containing 0.1% (v/v) triton X-100 and 1% goat serum and subsequently counterstained with 4',6-diamidino-2-phenylindole (DAPI; 13.0 ng/μL) in PBS, followed by two 5 min PBS washes. Coverslips were mounted with Hydromount and cells were imaged and captured using a Zeiss Axioskop fluorescent microscope and AxioCam HRm camera (Carl Zeiss) (Béraud et al., 2011 (link), 2013 (link); Béraud and Maguire-Zeiss, 2012 (link); Daniele et al., 2014 (link), 2015 (link)). Images and subsequent analyses were completed by an observer blinded to treatment.

We used monoclonal Antibodies against murine CD11b (M1/70), CD86 (GL-1), CD45 (104) from BD Biosciences, CD16/CD32 (2.4G2) from eBioscience for FACS analysis. Antibodies used for the OSC, spinal cord and brain histology: rat-MBP (1:500) from Millipore, rabbit-Iba1 (1:500) from WAKO Chemicals, mouse-Neurofilament (NF-M) (1:1000) from Convance Laboratories Inc., giunea pig-GFAP (1:1000) from SYnaptic Systems, rat-CD68 (1:500) from BioLegend, rat-Mac3 (1:500) from BioLegend, rat-TLR3 (1:500) from BioLegend, rb-pIRF7 (1:400) from Bioss-Antibodies and rabbit-CCR2 (1:500) from Bioss-Antibodies and rtLAMP2 (1:400) von BioLegend. A polyclonal crossreacting anti-GFP antibody was purchased from Abcam. Biotin conjugated donkey-anti-rabbit, as well as normal sera from mouse, rat and donkey were purchased from Jackson Immuno Research. All secondary antibodies conjugated with fluorophores (Cyanine Dye Cy2, Cy3 and Cy5) were purchased from Life Technologies and used in a dilution of 1 to 500.