Rabbit anti cd68

After the behavioural tests, the mice were anaesthetized and transcardially perfused with saline. Then, the brains were removed, and the hemispheres were separated. Brain samples assigned for immunohistochemical staining were immersed in OCT at -80 °C before coronal sectioning on a Frozen slicer (Thermo Fisher Scientific, USA) (10 μm thickness). The sections were restored to room temperature, fixed with cold acetone for 5 min, and permeabilizing agent was used for 5 min. Then, the cells were rinsed three times at room temperature for 5 min each with 1x PBS (pH 7.2-7.4) and blocked with 10% bovine serum in 0.1% Triton X-100 for 30 min. The blocking solution covered all brain tissues and prevented the tissue from drying out. Then, the samples were incubated with primary antibodies overnight at 4 °C. The primary antibodies included rabbit anti-NeuN (1:500, Abcam), rabbit anti-GFAP (1:200, CST), rabbit anti-Iba-1 (1:100, Abcam), rabbit anti-CD68 (1:300, Abcam), rabbit anti-APP (1:100, Millipore), and mouse anti-β-amyloid specific for Aβ₄₂ (1:200, CST). The next morning, the sections were incubated for 1 h at room temperature with DyLight 488-/555-conjugated goat anti-rabbit/mouse IgG (1:200, Abcam) and stained with DAPI for 3 min. Fluorescence images were captured using an Olympus FV3000 confocal microscope.

Synovial lining macrophages were quantified in liposome-treated joint tissue sections (n = 3 per condition) using immunofluorescence labelling as previously described [7 (link)]. Sections were labeled with rabbit anti-CD68 (Abcam) or rabbit polyclonal isotype control (Abcam, ab37415) at 0.005 mg/mL in blocking buffer overnight at 4^oC. Goat anti-rabbit-Alexa-488 secondary antibody (Jackson ImmunoResearch) at 0.0015 mg/mL was applied before washing and mounting with ProLong Gold Antifade Mountant with DAPI (Fisher Scientific). Slides were imaged on a Zeiss LSM 800 AiryScan confocal microscope using the 40X water immersion lens with configuration settings held constant across all samples. Two regions of interest were imaged per section.

The formalin-fixed liver tissue was processed, and 5-μm-thick paraffin sections were stained with haematoxylin and eosin (H&E) and Masson’s trichrome for histological analysis. The histological examination was performed using the histological scoring system for NAFLD by an experienced pathologist without prior knowledge of the treatments. The NAFLD activity score (NAS) was quantified by summing the scores of steatosis (0–3), lobular inflammation (0–2), and hepatocellular ballooning (0–2). NASH was defined in the cases of NAS of ≥547 (link)48 (link)49 (link). Immunohistochemistry approaches were performed as previously described50 (link), using a rabbit anti-CD68 (Abcam) and rabbit anti-α-SMA (Abcam).

Sections of paraffin-embedded spleen and lung tissues (4 μm in thickness) were prepared to detect antigen expression through immunohistochemistry (IHC) staining. Briefly, the retrieved sections were incubated overnight at 4 °C with the following antibodies: a mouse anti-C3 mAb (Hycult Biotech, The Netherlands), rabbit anti-C5b-9 (Calbiochem), and anti-C5aR (Santa Cruz Biotechnology, Paso Robles, CA, USA) polyclonal antibodies, rabbit anti-CD68 (Abcam, Cambridge, MA, USA) and anti-IFN-γRα (Santa Cruz Biotechnology) polyclonal antibodies, a rabbit cleaved caspase-3 Ab (Cell Signaling), a mouse anti-PCNA mAb (Santa Cruz Biotechnology), and a rabbit Novel coronavirus nucleoprotein/NP polyclonal Ab (Sino Biological Inc., Beijing, China). Biotinylated immunoglobulin G was then added, followed by an avidin–biotin–peroxidase conjugate (Beijing Zhongshan Biotechnology Co., Ltd.). Immunoreactivity was detected using 3,3' diamino benzidine (DAB) and by counterstaining with hematoxylin.