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.
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.
Free full text: Click here
