Dp70 camera system

OCT-embedded, cryopreserved mouse kidneys were cut into 7-μm sections, fixed with acetone, blocked with blocking buffer (1× PBS, 3% BSA, 0.5% Triton X-100) and stained with goat anti-mouse Podocin antibody (Santa Cruz Biotechnology; cat # sc-22296) (Supplementary Table 8) and then with Alexa Fluor 594-conjugated donkey anti-goat IgG (H+L) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (H+L) antibodies (Invitrogen; cat # A-21202). The stained sections were preserved in Vectashield antifade mounting medium with DAPI (Vector Laboratories; cat # H-1200-10) and imaged using an Olympus DP70 camera system attached to an IX70 fluorescence microscope (Olympus Life Science). A 200× magnification was used with 1/200 sec exposure for the green channel (IgG) and 1/50 sec exposure for the red channel (podocin). IgG IC deposition was scored blindly by the investigator following imaging.

For ALP staining, differentiated osteoblasts were washed with PBS and fixed in 10% formalin for 2 min. Cells were rinsed with deionized water and incubated with alkaline dye mixture for 30 min by wrapping with aluminum foil according to the manufacturer’s instruction (Sigma-Aldrich). Cells were then rinsed with deionized water and subjected to a microscopic observation. For alizarin red S staining, cells were washed with distilled water, fixed in ice-cold 70% ethanol for 1 h, and then stained with 2% Alizarin red S stain solution (adjusted to pH 4.2 with 10% ammonium hydroxide) (Sigma-Aldrich) for 30 min. Stained cells were washed and observed using a light microscope. At least 10 cell images were captured under a microscope (Olympus IX51 inverted fluorescent microscope, Olympus Optical, Japan) fitted with a DP70 camera system (Olympus) and used for a quantitative analysis with the Image J software. To determine ALP activity, cells were washed and lysed with Triton X-100 lysis buffer (50 mM Tris, 150 mM NaCl, and 1% Triton X-100, pH 10). Cell supernatants were incubated with an ALP substrate. ALP activity assay was performed in triplicate using the LabAssay ALP Kit (Wako Pure Chemical Industries, Japan). Relative ALP activity was calculated by comparing with standard solution and expressed as units per mg of protein according to the manufacturer’s instructions.

The GUS staining was performed as described by [28 (link)]. Cellular localization and uniform transformation of 35S:AtSTOP1:sGFP and 35S:CcMATE1:sGFP were analyzed based on sGFP fluorescence. The samples were observed and photographed under an Olympus BX51 microscope 9 (Olympus, Tokyo, Japan) equipped with Olympus DP70 Camera System (Olympus) and fluorescence microscope (AXIO imager system, Carl-Zeiss-Japan, Tokyo, Japan). The transgenic hairy roots were examined for the integration and co-integration of the transformed gene by genomic PCR.