Ab183597

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.

In order to estimate the adenoviral gene transfection and expression efficiency in vitro, HepG2 and 293 T cells were infected with four recombinant adenoviruses at multiplicity of infection (MOI) of 40 and 4, respectively. After incubation for 48 h, the GFP expression and infection efficiency were determined under fluorescence microscopy. The cell protein and growth medium were collected for Western blot and ELISA analyses. The protein of the transfected HepG2 and 293 T cells were extracted by a protein extraction kit (Bio Teke Corporation, China), and the protein concentration was determined by the BCA Protein Assay Kit (Bio Teke Corporation, China), according to the manufacturer’s protocol. Thereafter, total protein (15 µg) was loaded into each lane of acrylamide gel and subjected to SDS-PAGE. The membranes were incubated with antibodies against human ApoE (ab183597, Abcam) at 4 °C overnight; α-tubulin antibody (sc-8035, Santa Cruz Biotechnology) was used as an internal control to verify the basal expression level and equal protein loading. Target protein bands were captured by the chemiluminescence imager (iBright CL1000, Thermo Scientific).

WT and Apoe-homozygous knockout rats were generated by intercrossing Apoe-heterozygous knockout rats (F₁) and were subsequently sacrificed at six weeks of age to obtain tissue samples from the brain, liver, kidney, and spleen. Tissue lysates were prepared and analyzed by immunoblot using specific primary antibodies for apolipoprotein E (EPR19392, ab183597, Abcam, Cambridge, UK) and β-actin (C4, sc-47778, Santa Cruz, CA, USA).

Sections (3 μm) cut from 10% formalin-fixed, paraffin-embedded kidney specimens were immunostained for complement C9 (ab173302, Abcam, Cambridge, MA, USA) and ApoE (ab183597, Abcam), as previously described^{58 (link)}. Then the tissues were incubated with an HRP-conjugated goat anti-rabbit antibody (8114, CST, USA). Antibody binding was detected by incubation with a fresh mixture of diaminobenzidine (DAB, 8059S, CST, USA), according to the manufacturer’s instructions. Images were captured with a Nikon DXM 1200/NIS-Elements mounted on a light microscope (Nikon Eclipse E600, Shanghai, CHN) and analysed using Image J software. Complement C9 and ApoE staining in the glomeruli was quantified as ratio of staining-positive area/glomerular tuft area.