Ab95470

At the termination of different treatments, cells or spinal cord tissues (2.0 mm above and below the lesion, Supplementary Fig. 2) were collected to extract total proteins for western blot as the previously described (Wang et al. 2022 (link); Guo et al. 2020 (link)). In this study, the following specific antibodies were used: TG2 (3557, 1:1000, Cell Signaling Technology), PSR(PAB916Hu01, 1:1000, Cloud-Clone Corp.), iNOS (ab49999, 1:2000, Abcam), Arg-1 (ab60176, 1:3000, Abcam), CD86 (91882, 1:2000, Cell Signaling Technology), CD206 (60143-1-Ig, 1:2000, Proteintech), TREM2 (ab95470, 1:1500, Abcam), pNF-κB (3033, 1:2000, Cell Signaling Technology), APOE (ab183597, 1:2000, Abcam) and β-actin (T0022, 1:5000, Affinity). After primary antibodies overnight at 4 °C, the corresponding secondary antibodies were incubated based on the manufacturer’s instructions. The immunoblots were visualized using ECL kit and scanned by ChemiDoc XRS (Bio-Rad, CA, USA). Image J software was used to analyze the intensity of the bands based on the β-actin level.

Primary microglial cells were placed in in 24-well plate at the density of 20 × 10⁴ cells/well. After treatment with OGD and FTY720 incubation period, the cells were fixed in 4% paraformaldehyde solution at room temperature for 30 min and were blocked in 3% BSA containing 0.1% Triton X-100 for 1 h at room temperature. Then, the primary antibodies were used to incubate the samples overnight at 4°C in a humid chamber, and the Alexa Fluor secondary antibody were used to incubate the samples for 1 h at room temperature. The following antibodies were used: anti-CD86 Rabbit Polyclonal Antibody (1:100, #BS9900M, Bioworld), anti-CD206 Rabbit Polyclonal Antibody (1:500, #ab64693, abcam), anti-iNOS Mouse monoclonal antibody (1:200, #sc-7271, Santa Cruz Biotechnology Inc.,), anti-TREM2 Goat monoclonal antibody (1:500, #ab95470, abcam), goat anti mouse IgG ALEXA flour 488, donkey anti rabbit IgG ALEXA flour 546, donkey anti rabbit IgG ALEXA flour 488, and donkey anti goat IgG ALEXA flour 555 (1:1,000, #a11001, #a10040, #a21206, and #a21432, Invitrogen). After three more washed, stained nuclei were stained with Hoechst 33,342 for 20 min. Images were taken using a confocal microscope (Nikon A1RSi, Tokyo, Japan) and analyzed using Software ImageJ.

Sagittal sections from D0, D7, D30 knee joints of BL6 mice were used for IHC. Primary antibodies were incubated overnight at 4°C in a dark, humid chamber following antigen retrieval with Unitrieve (NB325 Innovex Biosciences, Richmond, CA. USA). Secondary antibodies were incubated for 2 hours at room temperature in a dark, humid chamber at 1:500. Negative control slides were incubated with secondary antibody-only. Stained slides were mounted with Prolong Gold with DAPI for nuclei staining (Molecular Probes, Eugene, OR. USA). Slides were imaged using a Leica DM5000 microscope. ImagePro Plus V7.0 Software, QIClick CCD camera (QImaging, Surrey, BC, Canada) and ImageJ V1.53 Software were used for imaging and photo editing. Primary antibodies include: Lyve1 [1:100 (5.5µg/mL); ab218535 Abcam, Cambridge, UK], Trem2 [1:100 (5.0µg/mL); ab95470 Abcam, Cambridge, UK], CD206 [1:100 (10µg/mL); 601431IG, ThermoFisher, Waltham, MA. USA]. Secondary Antibodies include Chicken anti-rabbit 594 (1:500; A21442, Thermofisher, Waltham, MA. USA), Chicken anti-Rabbit 488 (1:500; A21441, ThermoFisher, Waltham, MA. USA), Donkey anti-goat 488 (1:500; A11055, ThermoFisher, Waltham, MA. USA) and Goat anti-Mouse IgG2a 594 (1:500; A21135, ThermoFisher, Waltham, MA. USA).