Hrp conjugated antibodies

Cell lysates were prepared from frozen kidney tissue using RIPA Lysis and Extraction buffer (ThermoFisher Scientific) supplemented with a protease inhibitor cocktail (Merck). Proteins were separated on pre-casted 4–12% SDS page gels (ThermoFisher Scientific) before being transferred to PVDF membranes. Blots were blocked in 5% skimmed milk before being incubated with primary antibodies specific for CCL17 or alpha-tubulin followed by secondary HRP-conjugated antibodies (Abcam), according to manufacturer’s recommendations. The membranes were developed with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) and the results imaged by Bio-Rad Universal Hood II Gel Doc XR System. The protein levels were quantified by ImageJ (51 (link)).

Western Blot analysis was performed on neuronal cell lysates following pharmacological treatments with BSO and Diamide. Protein levels were normalized using a Bradford assay and resolved by SDS–PAGE, transferred to polyvinylidene difluoride membrane and probed with the following antibodies: Drp1 (Abcam ab56788), Mitofusin 1 (Abcam ab57602), Mitofusin 2 (Abcam ab56889), Opa1 (Abcam ab42364) and GAPDH (Abcam ab9485). Bands were visualized using Hrp conjugated antibodies (Abcam). 3 samples were used per group and experiments were run in triplicate.

Histological staining for immunohistochemistry was performed on heart tissue that had been harvested, fixed in 4% paraformaldehyde, embedded in paraffin, sliced into sections, and blocked with 5% BSA for 0.5 h, after which the sections were washed with PBS. The samples were then covered with 5% BSA for 0.5 h and incubated with antibodies (anti-CD31, 1 : 200; Abcam, anti-Arg-1, 1 : 100; ProteinTech Group, anti-CD206 1 : 100; ProteinTech Group) at 4°C overnight. For histochemistry, the samples were then immunostained with HRP-conjugated antibodies (Abcam) for 4 h at room temperature. For immunofluorescence, after the tissue incubated with antibody overnight and then washed three times with PBS, the sections were incubated with appropriate secondary antibody Alexa Fluor 594 AffiniPure donkey anti-rat for 2 hours at room temperature. After 3 times additional washing with PBS, the sections were sealed with DAPI Fluoromount-GTM (Yeasen) to identify nuclei. Fluorescence microscopy images were caught with microscope (IX83; Zeiss). Masson's trichrome staining was performed for cardiac fibrosis analysis. Staining was observed using fluorescence microscope (IX83; Zeiss). Quantification of all data was performed with the ImageJ software.2.2.