Oct media

Drugs utilized in this study were obtained as follows – rTM (Asahi Kasei Pharma Co., Tokyo, Japan); and sCR1 (CDX-1135; Celldex Therapeutics, MA, USA); αCD47Ab (murine-specific: MIAP 301 [sc-12731]; porcine-specific: BRIC-126 [sc-59079]; Santa Cruz Biotechnology, TX, USA).
Other major reagents were obtained from the following sources – RNAlater solution (Ambion/Thermo Fisher Scientific, TX, USA); OCT media (Tissue-Tek, ProSciTech, Australia); Bond Polymer Refine Detection Kit (Leica Biosystems, Newcastle upon Tyne, UK); immunofluorescence mounting media (Dako/Agilent Technologies, CA, USA); rat anti-mouse Ly-6G/Ly-6C antibody (RB6-8C5) (Biolegend, CA, USA); rabbit anti-rat IgG (BA-4001) (Vector Laboratories, CA, USA); RNA extraction kit (Bioline/Meridian Life Science, TN, USA); gene-specific primers (Thermo Fisher Scientific, MA, USA); complement C3 antibody (Thermo Fisher Scientific);vC9 polyclonal antibodies (Abcam, Cambridge, UK); goat anti-rabbit Alexa Fluor 647 secondary antibody (Thermo Fisher Scientific); TUNEL staining kit (In Situ Cell Death Detection Kit, TMR Red; Sigma-Aldrich/Merck, MO, USA); University of Wisconsin (UW) solution (Bridge to Life, SC, USA); creatinine (Merck, Darmstadt, Germany); and DHE (Thermo Fisher Scientific)

Transcardial perfusion and tissue fixation procedures were performed as previously described [46 (link)]. Briefly, the mouse was terminally anesthetized with sodium pentobarbitone (80 mg/kg i.p.), and their spleens were extracted and weighed. The mouse underwent transcardial perfusion with saline (3 mL/min for 5 min) followed by 4% paraformaldehyde. The whole brain was collected, post-fixed in 4% paraformaldehyde overnight and then immersed in 30% sucrose for 3–5 days before being embedded in Optimal Cutting Temperature (OCT) media (ProSciTech, Kirwan, Australia) for sectioning.