Dark field microscope

Immunofluorescence staining was performed on O.C.T-embedded frozen kidneys, which were sectioned at 8 µm before staining. Kidney sections were fixed in 4% PFA (Fisher Scientific, Waltham, MA, USA) before staining. Primary antibodies of thrombin, CD31, VCAM-1, NLRP3, Caspase-11, IL-1beta, or IL-18 (Abcam, Waltham, Boston, MA, USA) were applied onto the frozen section for 1 h at room temperature, followed by the corresponding secondary antibodies against rabbit labeled with Alex 594 or Cy5.5 (Abcam, Waltham, Boston, USA) incubation at room temperature for 30 min. Slides were then mounted with VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, Newark, CA, USA) before being imaged with an Olympus dark-field microscope outfitted with a HAMAMATSU digital camera C11440 at 20× or 40× magnification. Double-blind data acquisition was performed on all images.

10% formalin-fixed kidneys were further processed, sectioned, and H&E stained at the Anatomic and Molecular Pathology Core Histology Lab of the Department of Pathology and Immunology, Washington University School of Medicine. Microscopic images of the H&E slides were acquired with an Olympus bright-field microscope outfitted with a DP27 digital camera to record images of the tissue sections at 20× magnification.
Immunofluorescence staining was performed on O.C.T-embedded frozen kidneys, which were sectioned at 8 µm before staining. Kidney sections were fixed in 4% PFA (Thermo Scientific, Waltham, MA, USA) before staining. Primary rabbit antibodies to p62, ICAM-1, VCAMM-1, and Bax (Abcam, Waltham, MA, USA) were applied onto the frozen section for 1 h at room temperature, followed by the secondary anti-rabbit antibody labeled with Alex 594 (Abcam, Waltham, MA, USA), and the tissue section was incubated at room temperature for 30 min. Slides were then mounted using VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, Newark, CA, USA), before imaging with an Olympus dark-field microscope outfitted with a HAMAMATSU digital camera C11440 at 40× magnification. Double-blind data acquisition was performed on all images. Blinded data analysis was performed on all kidney images by an experienced renal pathologist (J.G.).