Anti stat3

Formaldehyde-fixed aortic tissue was incubated in PBS overnight at 4°C. Then, it was embedded in an OCT compound (Tissue-Tek) and serially sectioned on a cryostat. Serial 8 μM sections were stained with HE staining and immunofluorescence. For immunofluorescence, paraffin-embedded sections were deparaffinised with xylene and rehydrated through graded ethanol and then incubated in 10 mmol/l citrate buffer for antigen retrieval. Primary antibodies (1 : 200, anti-JAK2 (abcam, USA); 1 : 100, anti-STAT3 (Bioworld, China); 1 : 200) were used and then incubation of samples were treated with anti-rat Alexa Fluor 488 secondary antibody (abcam, USA) for 30 min at 37°C. Afterwards, the samples were incubated with DAPI solution for 5 min in the dark; they were washed 3 times with PBS and examined under a fluorescence microscope. Then, cells were plated on autoclaved glass coverslips placed in sterile 6-well plates, fixed in 4% paraformaldehyde, permeabilised in 0.1% Triton, and prevented with 10% goat serum. Subsequently, cells were treated with primary antibodies (1 : 200, anti-JAK2 (abcam, USA); 1 : 100, anti-STAT3 (Bioworld, China)) for 1 h at 37°C. After rinsing with PBS 3 times, the fluorescent secondary antibodies were added and the cells were stained as detailed above.

Proteins were extracted from mouse kidney cortex and cultured cells with RIPA lysis buffer (Beyotime, Jiangsu, China). Protein concentration was measured by BCA Protein Assay kit (Bio‐Rad, Hercules, CA, USA). After boiling for 5 min. at 95°C in 5× loading buffer, an equal amount of protein (40 μg) was separated via 8% SDS‐PAGE and electrotransferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). The membranes were routinely processed via blocking with 5% milk or 5% BSA and were incubated overnight with primary antibodies, followed by a 1‐hr incubation with horseradish peroxidase‐conjugated secondary antibodies. Immunoreactive proteins were detected using an Enhanced Chemiluminescence (ECL) Kit (Amersham). The antibodies used in this study were as follows: anti‐mIL‐6R (rabbit, 1:200), anti‐gp130 (rabbit, 1:1000), anti‐phospho‐STAT3 Tyr705 (mouse, 1:200) and β‐actin (mouse, 1:10,000) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐sIL‐6R (mouse, 1:500; Abcam, Cambridge, MA, UK); and anti‐desmin (rabbit, 1:1000), anti‐phospho‐STAT3 Ser727 (rabbit, 1:1000) and anti‐STAT3 (rabbit, 1:1000) from Bioworld Technology (Louis Park, MN, USA). Densities of blots were measured by ImageJ software (NIH, Bethesda, MD, USA).