Alexa fluor 594 conjugated igg

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).

The eyecups were fixed with 4% paraformaldehyde and embedded in optimal cutting temperature compound. Frozen sections of 10 μm thickness were fixed in cold acetone (−20°C) for 10 min. Sections were incubated with 0.2% Triton X-100 for 20 min and blocked with 2% BSA in PBS for 60 min. Then, sections were incubated with different primary antibodies for 16 h at 4°C. After washing three times, sections were incubated with Alexa Fluor 594-conjugated IgG (Abcam) or Alexa Fluor 488-conjugated IgG (Abcam) for 60 min at 37°C. The nucleus was counterstained with 4′, 6-diabmidino-2-phenylindole (Abcam). Images were acquired using a Leica microscope system (DM2500, Wetzlar, Germany).