Galectin 3

The fixation of tissue sections was performed with 4% paraformaldehyde in PBS for 5 min at room temperature. Following fixation, the sections were washed at room temperature with PBS containing 0.1% Triton X-100 for 5 min. To block nonspecific antibody binding, the sections were pretreated with PBS containing 5% goat serum for 30 min at room temperature. Subsequently, primary antibodies (vimentin, galectin-3, N-cadherin, and E-cadherin) were applied to the slides and incubated at room temperature for 1 h. The primary antibody dilutions used were as follows: vimentin (1:1000, Abcam), galectin-3 (1:1000, Abcam), N-cadherin (1:1000, Abcam), and E-cadherin (1:1000, Abcam). After rinsing, the sections were incubated with HRP-labelled goat anti-rabbit IgG secondary antibody (ab6721) for 1 h at room temperature.

After harvesting, kidneys were immediately fixed in 4% paraformaldehyde and then embedded in paraffin. The tissues were sectioned and stained with hematoxylin & eosin (H&E) stain, periodic acid Schiff (PAS) stain, and Masson’s trichrome stain. Images were captured using a confocal microscope (Nikon, Tokyo, Japan). Tubular injury in PAS-stained sections was assessed and scored at a ×200 magnification using 10 randomly selected fields for each kidney as follows: 0, 0%; 1, ≤10%; 2, 11–25%; 3, 26–45%; 4, 46–75%; and 5, 76–100% [18 (link)]. For IHC staining, the sections were incubated with primary antibodies against kidney injury molecule-1 (Kim-1; Abcam, Cambridge, MA, USA), neutrophil gelatinase-associated lipocalin (NGAL; Santa Cruz Biotechnology, Santa Cruz, CA, USA), 4-hydroxynonenal (4-HNE; Abcam), galectin-3 (Abcam), α-smooth muscle actin (α-SMA; Sigma-Aldrich), or collagen I (Abcam) overnight at 4 °C and then probed with a secondary antibody for 30 min at room temperature. All the sections were counterstained with hematoxylin. The percentage of positive staining was assessed in 5 randomly selected fields (×400 magnification) per each kidney using i-Solution Lite V.9.1 image analysis software (IMTechnology, Vancouver, BC, Canada). Galectin 3 positive cells or CD4 positive cells were counted in 5 randomly selected fields (×400 magnification) per each kidney.

To detect the expression of Cytokeratin 12 (CK12), galectin-3, and CD11b in fungal keratitis, immunofluorescence staining in mouse corneas of fungal keratitis was performed. Corneal tissues were fixed with 4% paraformaldehyde for 10 minutes. The mouse cornea tissues were then equilibrated in 30% sucrose solution overnight for cryoprotection, embedded in OCT media, and sectioned using a Cryostat set at 10 μm per section. Slides of corneal tissues were saturated with 0.5% Triton X-100 in PBS for 60 minutes and blocked with 3% BSA for 30 minutes. The slides and cells were incubated with primary antibodies specific for CK12 (1 : 200, Abcam, UK), galectin-3 (1 : 200, Abcam, UK), and CD11b (1 : 500, Abcam, UK) for one hour at room temperature. After washing 5 minutes, 3 times in PBS, slides were incubated with respective secondary antibodies of Alexa Fluor 594-conjugated IgG (1 : 300, Abcam, UK) or Alexa Fluor 488-conjugated IgG (1 : 300, Abcam, UK) for one hour at room temperature, and the nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 minutes. Fluorescence images were acquired by a laser confocal fluorescence microscope (LSM800; Carl Zeiss Microscopy, White Plains, NY, USA).