Alexafluor 647 conjugated goat anti rabbit igg antibody

Neutrophils grown on coverslips in the lower chamber were incubated in 1× blocking buffer (5% bovine serum albumin in PBS) for 30 minutes. Cells were stained with an anti‐neutrophil elastase primary antibody (1:200; Abcam) in blocking buffer for 2 hours at room temperature, and then an AlexaFluor 647‐conjugated goat anti‐rabbit IgG antibody (1:200; Abcam) for 1 hour in the dark at room temperature. Then, the cells were fixed with 4% PFA for 30 minutes, permeabilized with 0.5% Triton X‐100 in phosphate‐buffered saline (PBS) for 30 minutes, and incubated with primary antibodies against histone H3 (1:200; Abcam) overnight at 4°C, followed by incubation with an AlexaFluor 488‐conjugated goat anti‐rabbit IgG antibody (1:200; Abcam) for 2 hours in the dark at room temperature. DAPI was used to stain DNA. Images were acquired using a confocal microscope (Zeiss LSM 510 Meta, Carl Zeiss, Jena, Germany).

Mitochondrial DNA (5 μg/mouse) was injected into mice through the tail veins, and 2 hours later, the lung tissues were removed and frozen for examination. The frozen lung tissues were cut into 5‐μm‐thick sections, which were incubated in acetone at 4°C for fixation. Then, the sections were incubated in 1× blocking buffer (5% bovine serum albumin in PBS) for 30 minutes and stained with an anti‐neutrophil elastase (1:200; Abcam) primary antibody in blocking buffer using for 2 hours at 37°C, and then an AlexaFluor 647‐conjugated goat anti‐rabbit IgG antibody (1:200; Abcam) for 30 minutes at 37°C in the dark. The sections were permeabilized using 0.5% Triton X‐100 and for 30 minutes and incubated with a primary antibody against histone H3 (1:200; Abcam) overnight at 4°C, followed by incubation with an AlexaFluor 488‐conjugated goat anti‐rabbit IgG antibody (1:200; Abcam) for 30 minutes at 37°C in the dark. DAPI was used to stain DNA. Images were acquired using a confocal microscope (Zeiss LSM 510 Meta).

Multicolor deep imaging was performed as described previously [15 (link)]. Tissue clearing and stain the peritoneal surface cells were performed by ScaleSQ (0) method [31 (link)] and intraperitoneal injection of 1,10-Dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate (DiI) [15 (link)], respectively. Nuclei were stained by following as previously [30 (link)]. Clearing and staining tissue was observed by inverted confocal microscope (LSM 710 with spectral imaging equipment, Carl Zeiss Microimaging GmbH, Jena, Germany). The acquisition software was ZEN 2012. Objective lenses were a 20× dry lens (EC Plan-Neofluar, numerical aperture (NA): 0.5; working distance 17(WD): 2.0 mm) and 40× oil-immersion lens (EC Plan-Neofluar, NA: 1.30 WD: 0.21 mm).
To identification of transgene-positive cell, we conducted the 3 dimensional (3D)-immunohistochemistry by CUBIC method [32 (link)]. To observe the mesothelial cells, we used a 1:450 dilution of anti-mesothelin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as primary antibody and a 1:750 dilution of Alexa Fluor® 647-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, MA, USA) as secondly antibody.