Cy3 labeled donkey anti rabbit igg

The abdominal aortas and kidneys of rats were removed and fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 day at 4°C, dehydrated through a graded ethanol series, and embedded in paraffin. The sections were cut with a microtome (SM 2000 R, Leica Biosystems, Wetzlar, Germany) and placed on PLATINUM PRO slides (Matsunami, Osaka, Japan) as previously described [35 (link)]. For observation of the renal glomerular basement membranes, kidney sections (1 μm thick) were deparaffinized and stained with periodic acid methenamine silver (PAM). For immunohistochemistry, deparaffinized aorta sections were treated for antigen retrieval by heating in an autoclave in 1 mM EDTA at 121°C for 5 min, and were then incubated overnight at 25°C with mouse anti-α-smooth muscle (SMA) (1:200; A5228, Sigma) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin. After washing with PBS, the sections were incubated with Cy3-labeled donkey anti-rabbit IgG, Alexa Fluor 488-labeled donkey anti-mouse IgG (Jackson Immunoresearch, West Grove, PA, USA), and 4’,6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan) for 2 h at room temperature. The specimens were examined with a BX53 microscope equipped with a DP80 microscope digital camera and cellSens imaging software (Olympus Optical, Tokyo, Japan).

Anesthetized mice were transcardially perfused with 0.9% saline. Mouse brains were removed and fixed in 4% paraformaldehyde for 48 h. Fixed brains were cryopreserved in 30% sucrose in PBS for at least 2 days, and coronal brain sections (30 μm) were obtained with a sliding microtome. For immunostaining, floating brain sections were permeabilized and incubated in blocking solution (10% normal goat serum in 0.3% Triton X-100 TBST) at room temperature for 1 h. Sections were then incubated with primary antibodies, including MC1 (1:500), acH3K18 (1: 1000) and Dapi. After overnight incubation, the sections were incubated with secondary antibodies including Cy3-labeled donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch) and fluorescein-labeled goat anti-mouse IgG (1:500, Vector Laboratories). Images were acquired by Keyence BZ-X7000 microscope, and analyzed using ImageJ software (NIH). Experimenters quantifying immunoreactivity were blind to the mouse genotype and treatment conditions.

Immunohistochemistry was performed, as described in our previous report [32 (link)]. Briefly, coronal sections (12 µm) were cut in a cryostat and mounted on gelatin-coated slides. The sections were blocked in 1% BSA, 5% normal donkey serum, and 0.20% Triton X-100 (pH 7.4) for 1 h at room temperature. Next, the sections were stained with antibodies specific to KL at 4 °C overnight. Primary antibodies were visualized with Cy3-labeled donkey anti-rabbit IgG (Jackson Lab). Images were captured with a Zeiss LSM800 confocal microscope.

The kidneys, lungs, and liver of each mouse were rapidly frozen in liquid nitrogen and embedded in OCT compound (Sakura, Tokyo, Japan). The embedded organ samples were sectioned at 10 μm thickness. The organ sections were then fixed with cold acetone for 10 min, followed by incubation in a blocking buffer (CAS Block, Thermo Fisher Scientific, Wilmington, DE, USA) at 25 °C for 1 h. Subsequently, they were incubated at 4 °C overnight with mouse anti-β-catenin (1:200) and rabbit anti-NF-κB (1:200) antibodies in phosphate-buffered saline (PBS). Next, the sections were washed three times with PBS containing 0.03% Triton X-100 and then incubated with Cy3-labeled donkey anti-rabbit IgG (1:400; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and FITC-labeled donkey anti-mouse IgG (1:400; Jackson ImmunoResearch Laboratories) antibodies in PBS at 25 °C for 1 h. After counterstaining with 1 μg/mL of DAPI (Sigma-Aldrich), the sections were examined using a confocal microscope (Nikon, Tokyo, Japan).