Anti iba1 antibody

Lumbar spine tissues were freshly isolated and paraffin-embedded after fixation with 10% neutral buffered formalin. Immunofluorescence was performed on paraffin-embedded sections (5 μm). After preparation of the slides as described previously [22 (link)], tissue samples were incubated with anti-IBA1 antibody (Cell Signaling, 17198, Danvers, MA, USA) at 4 °C overnight. Samples were then incubated with goat anti-rabbit Alexa Flour 594 (Invitrogen, A32740, Carlsbad, CA, USA) for 1 h at room temperature. Tissues were mounted with Slow Fade (Invitrogen, s2828, Carlsbad, CA, USA), followed by a coverslip, and the edges were sealed to prevent drying. Specimens were examined with Zeiss laser scanning microscope (LSM) 710. Fluorescence intensity was determined by using Image J software, Image J Version Fiji, https://imagej.net/software/fiji/, accessed on 10 November 2022). This method determines the corrected total fluorescence by subtracting out background signals, which is useful for comparing the fluorescence intensity between cells or regions.

Cells were lysed and total protein was followingly extracted by RIPA lysis buffer (Beyotime), with the concentration evaluated by a BCA protein assay kit (Beyotime). Protein samples were followingly mixed with loading buffer, denatured, and separated by SDS‐PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA, USA), which was blocked with 5% skimmed milk for 1 h at ambient temperature and incubated with the corresponding primary antibodies: anti‐Iba‐1 antibody (17198; Cell Signaling Technology, MA, USA), anti‐β‐actin antibody (8457; Cell Signaling Technology), anti‐STING antibody (13647; Cell Signaling Technology), anti‐phospho‐TANK binding kinase 1 (TBK1) antibody (5483; Cell Signaling Technology), and anti‐TBK1 antibody (3504; Cell Signaling Technology) overnight at 4°C, followed by incubation with HRP‐conjugated secondary antibody (Beyotime) for 1.5 h at ambient temperature. The protein bands were developed with an enhanced chemiluminescence kit (Beyotime). Ultimately, the relative expressions of proteins were analyzed by Image J software (NIH, USA) with β‐actin as the internal reference.

Brain tissues were homogenized in RIPA buffer (MilliporeSigma) containing complete protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche). After centrifugation (100,000g for 1 hour at 4°C), the supernatant was used for biochemical analysis as described (53 (link)). ApoE (53 (link), 55 (link)), collagen IV (LS-F20750, LSBio), MMP-2 (MMP200, R&D Systems), and MMP-9 (NBP2-60095, Novus Biologicals) in brain lysate were measured by ELISA. Brain lysate measurements were normalized to total protein concentrations determined by BCA assay (Thermo Fisher Scientific). Total cholesterol and triglycerides in plasma were measured using a Cholesterol Assay Kit (A12216, Thermo Fisher Scientific) and Triglyceride Assay Kit (ab65336, Abcam), respectively. Some brain samples were subjected to Western blotting using anti–Iba-1 antibody (17198, Cell Signaling Technology) and anti–β-actin antibody (3700, Cell Signaling Technology), followed by quantification through LI-COR Odyssey.