Anti pgc 1α antibody

Paraffin-embedded kidney tissue sectioned at 3-μm thickness was deparaffinized in xylene and rehydrated in a graded ethanol series to phosphate-buffered saline. 8-Hydroxydeoxyguanosine (8-OHdG) is a DNA oxidation product that was measured to assess DNA damage. Nutrient sensing signal AMPKα2 and PGC-1α were also analyzed by immunohistochemistry. Following blocking with immunoblock (BIOTnA Biotech., Kaohsiung, Taiwan), the sections were incubated for 2 hr at room temperature with an anti-8-OHdG antibody (1:100, JaICA, Shizuoka, Japan), an anti-AMPKα2 antibody (1:400, Proteintech, Rosemont, IL, USA), and an anti-PGC-1α antibody (1:200, Abcam, Cambridge, MA, USA). Immunoreactivity was revealed using the polymer-horseradish peroxidase (HRP) labelling kit (BIOTnA Biotech). For the substrate-chromogen reaction, 3,3′-diaminobenzidine (DAB) was used. Identical staining protocol omitting incubation with primary antibody was employed to prepare samples that were used as negative controls. Quantitative analysis of positive cells per microscopic field (X400) in the renal sections was performed as we described previously [7 (link)].

Western blot analysis was performed as our article described previously [4 (link)]. Total proteins from the BMSCs, MLE-12 cells, and lung tissues were lysed by the RIPA buffer (Beyotime, P0013B), and protein levels were measured with a BCA assay. The protein lysates (30 μg protein) were separated by 8%-12% SDS-PAGE and transferred onto the PVDF membrane. The membranes were blocked with 5% nonfat milk. After washing with TBST three times, the membranes were incubated with anti-CD63 antibody (1 : 1000; CST, USA), anti-TSG101 antibody (1 : 1000; CST, USA), anti- Nrf1 antibody (1 : 1000; Abcam, USA), anti-Tfam antibody (1 : 1000; Abcam, USA), anti-PGC-1α antibody (1 : 1000; Abcam, USA), anti-p-Drp1 antibody (1 : 1000; CST, USA), and anti-β-actin antibody (1 : 10000; CST, USA) overnight at 4°C. After washing with TBST, the membranes were again incubated with a secondary antibody for 1 h at room temperature. Finally, the membranes were visualized with a chemiluminescence system. The expression of proteins was normalized to β-actin as a reference.

Paraffin-embedded tissues sectioned at 3-μm thickness were deparaffinized in xylene and rehydrated in a graded ethanol series to phosphate-buffered saline. Following blocking with immunoblock (BIOTnA Biotech., Kaohsiung, Taiwan), the sections were incubated for 2 h at room temperature with an anti-phosphorylated AMPKα2 antibody (1:400, Cell Signaling, Danvers, MA, USA) or an anti-PGC-1α antibody (1:200, Abcam, Cambridge, MA, USA). Immunoreactivity was revealed using the polymer-horseradish peroxidase (HRP) labeling kit (BIOTnA Biotech). For the substrate–chromogen reaction, 3,30-diaminobenzidine (DAB) was used. An identical staining protocol omitting incubation with primary antibody was employed to prepare samples that were used as negative controls. Renal cells positive for immunostaining were examined in 10 randomly selected ×400 microscopic fields per section. The number of immunostained cells was expressed as we described previously [18 (link)].