Anti rabbit igg

The serum was sent to the clinical chemistry laboratory at Srinagarind Hospital, Khon Kaen, Thailand. The estimated glomerular filtration rate (eGFR) was calculated based on the CKD-EPI equation (Levey et al., 2009 (link)). Urine samples were stored at −80°C before protein detection. We developed an in-house indirect enzyme-linked immunosorbent assay (ELISA) to detect ferritin protein in urine. Urine from a random selection of 148 samples, diluted at 1:10, was plated overnight at 4°C on 96-well flat-bottom, high-binding plates without lids (Corning, United States of America). After blocking with 5% skimmed milk, the primary antibody, diluted at 1:500 anti-ferritin light chain rabbit monoclonal antibody (A11241, Abclonal, MA, United States of America), was added and incubated for 1 h at 37°C. After washing, the secondary antibody (Anti-rabbit IgG, HRP-linked antibody; diluted at 1:500) (7074S, ABclonal, MA, United States of America) was added and incubated again at 37°C for 1 h. O-phenylenediamine dihydrochloride (OPD) (Thermo Scientific) was added to each well. The optical density (OD) of the wells was read at the wavelength of 492 nm using a microplate reader (Sunrise, Tecan Trading AG, Switzerland).

Nitroglycerin injection was purchased from Beijing Yimin Pharmaceutical Co., Ltd. (Beijing, China). DMSO, PBS and sodium dodecyl sulfate polyacrylamide gel kits were obtained from Solarbio (Beijing, China). Primary antibodies, including anti-c-Fos, anti-NF-κB p65, anti-NF-κB p-p65, anti-RAGE, anti-LRP1, anti-AQP4, anti-MFSD2A, anti-ZO-1, anti-Occludin, anti-VE-cadherin-2, anti-β-actin, and anti-Iba1, were furnished by Cell Signaling Technology (Beverly, MA, USA). anti-iNOS and anti-IL-17A antibodies were purchased from Abcam (Shanghai, China). Anti-fitc-CD₄, Anti-alexa fluor 488-rabbit IgG, anti-cy3-mouse IgG, anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from ABclonal (Wuhan, China).

Mdivi-1(mitochondrial division inhibitor-1) and TTC (2,3,5-triphenyltetrazolium chloride) were purchased from Sigma Aldrich (St. Louis, Missouri, MO, USA); BSA (bovine serum protein) was obtained from BOSTER (Boster Biological Technology, Wuhan, China, AR1006). Blots were incubated with antibodies against ATF4 (Proteintech, Rosemont, Pennsylvania, USA, 10835-1-AP), Parkin (CST, Boston, Massachusetts, USA, #4211), TOM20 (Proteintech, USA, 11802-1-AP), COX4I1 (Proteintech, USA, 60251-1-Ig), NLRP3 inflammasome (Affinity, Cincinnati, Ohio, USA, DF7438), pro-caspase-1 (ABclonal, Wuhan, China, A0964), cleaved caspase-1 (CST, USA, #67314), pro-IL-1β (ABclonal, China, A11370), cleaved IL-1β (Affinity, USA, AF4006), pro-IL-18 (Proteintech, USA, 10663-1-AP), cleaved IL-18 (R&D Systems, Minneapolis, Minnesota, USA, AF521) and β-actin (ABclonal, China, AC004), followed by the secondary antibodies conjugated to horseradish peroxidase anti-rabbit IgG (H + L) (ABclonal, China, AS014) and anti-mouse IgG (H + L) (ABclonal, China, AS003).
The primary antibodies used for the immunofluorescence analysis were against TOM20 (Proteintech, USA, 11802-1-AP) and COX4I1 proteins (Proteintech, USA, 60251-1-Ig). Brain sections were then incubated with the secondary antibodies goat anti-rabbit DyLight 488 (Abbkine, green, A23240) and goat anti-mouse DyLight 549 (Abbkine, red, A23310).