Ab96762

Kidney sections (4 µm) were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked using 0.3% H₂O₂ at 37 °C for 10 min. Heat-mediated antigen retrieval was performed using 10% citrate buffer. Then the sections were incubated with primary antibodies followed by secondary antibody and visualized with diaminobenzydine (DAB). Primary antibodies and dilutions used were as follows: Col IV (1:200; ab6586, Abcam), SNRK (1:500; ab96762, Abcam). The stained slides were photographed using an OLYMPUS BX51 microscope and the percentages of positive cells and staining intensities were scored as previously described^{58 (link)}.

Mouse and human tissues were collected and fixed with 3.7% paraformaldehyde (v/v)^{43 (link),54 (link),55 (link)}. Briefly, deparaffinized sections were treated with proteinase-K (25 µg mL⁻¹ for 8 min at 37 °C). Double-FAM-labeled miR-103a-3p probe (mmu-miR-103a-3p miRCURY LNA miRNA Detection probe, Cat YD00615519, Exiqon, Denmark) was incubated at 30 nM for 1 h in Exiqon hybridization buffer (Exiqon) at 57 °C. Polyclonal sheep anti-vWF (ab11713, 1:100, Abcam, Cambridge, UK) was incubated at room temperature and detected with Alexa Fluor 555-conjugated donkey anti-Sheep (1:400, Thermo Fisher Scientific, Waltham, MA). And polyclonal Rabbit anti-SNRK (1:100, ab96762, Abcam, Cambridge, UK) was incubated at room temperature and detected with Alexa Fluor 488-conjugated goat anti-Rabbit (1: 400, Thermo Fisher Scientific, Waltham, MA). Slides were mounted with Anti-fade Gold with DAPI (Invitrogen). Image acquisition was performed with confocal microscope (LSM800, Carl Zeiss Microscopy Ltd, Cambridge, MA). The colocalization coefficient was quantified using Zen 2011 software. For each channel, the colocalization coefficient was calculated as the ratio of pixels exhibiting colocalized SNRK to total pixels exhibiting vWF staining. All quantitation was done using original, unmodified data images.

Western blotting was performed as previously described^{59 (link)}. Proteins were extracted from cells/tissue using RIPA lysis buffer. The protein concentrations were assayed by bicinchoninic acid method. Equal amounts of protein (20–40 μg) was separated by SDS-PAGE and then transferred electrophoretically to polyvinylidene fluoride membranes. Membrane was incubated with a 1:1000 dilution of primary antibody at 4 °C overnight, followed by a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized by enhanced chemoluminescence system. Primary antibodies and dilutions used were as follows: SNRK (1:1000; ab96762, Abcam), MCP-1 (1:1000; ab7202, Abcam), TNF-α (1;1000; 3703, Cell Signal Technology), Col I (1:1000; 84336, Cell Signal Technology), and Col IV (1:200; ab6586, Abcam). GAPDH was used as an internal reference. The image collection and quantitative analysis were performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA USA). Uncropped immunoblot gels are displayed in Supplementary Fig. 19.