Protein blocker

For Immunohistochemical analysis, kidney sections were deparaffinized and rehydrated, and to expose the antigens, they were boiled in a target retrieval solution (TRIS buffer pH 9.0) for 30 min. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 15 min at room temperature. Nonspecific binding was prevented by incubating the sections with a protein blocker (Dako, CA). Sections were incubated overnight at 4°C with primary rabbit anti‐caspase‐3 active monoclonal antibody (1:100, Santa Cruz Biotechnology, CA). After washing with TBS, the sections were incubated with horseradish peroxidase‐conjugated polymer (Dako) for 30 min at room temperature. Slides were rinsed with PBS, and the antibody–antigen reactions were visualized with 3, 3’‐diaminobenzidine (Dako, CA). The sections were lightly counterstained with hematoxylin. Analyses were performed using light microscopy (Leica Imaging Systems) at 20× magnification, and the stained proteins were quantified using Corel Photo‐Paint 12 and UTHSCSA – Image Tool software. The picture analysis was performed in two phases: first, measurement of the tissue area with the subtraction of the blank areas on the picture; second, calculating the percentage of staining present on the first phase picture.

Femoral bones were cryoembedded in OCT compound and sectioned at 8-µm thickness. Sections were fixed with methanol for 15 min and blocked with a protein blocker (Dako). Specimens were then incubated with goat anti–VE-cadherin antibody (R&D Systems) followed by donkey anti–goat IgG antibody conjugated to Alexa Fluor 488 (Molecular Probes) and nuclear staining. Images were acquired with a confocal laser-scanning microscope (FV1000; Olympus). Blood vessel perimeters were measured using ImageJ (NIH).

The kidney slices were deparaffinized and rehydrated, as described above. To expose the antigens, the kidney sections were boiled in a target retrieval solution [citrate buffer (pH 6.0) for fibroblast-specific protein 1 (FSP1), TRIS buffer (pH 9.0) for α-smooth muscle actin (α-SMA) and ED-1] for 30 min. Endogenous peroxidase activity was blocked with 3% H₂O₂ (Labsynth) for 10 min at room temperature. Nonspecific binding was prevented by incubating the sections with a protein blocker (Dako, Carpinteria, CA, USA). The sections were incubated overnight at 4°C with primary antibodies: α-SMA (cat. no. A2547; 1:500; Sigma-Aldrich), FSP1 (cat. no. A5114; 1:400; Dako) or CD68/ED-1 (cat. no. MCA341R; 1:100; Serotec, Oxford, UK). Following washing with Tris-buffered saline (TBS) [50 mM TRIS, 150 mM NaCl], the sections were incubated with a horseradish peroxidase-conjugated polymer (Dako) for 30 min at room temperature. The slides were rinsed with TBS and the sites of antibody-antigen binding were visualized with 3,3′-diaminobenzidine (Dako). The sections were lightly counterstained with hematoxylin. The analyses were performed using light microscopy (Eclipse 2000 camera Nikon DS-Fi2) and the stained proteins were quantified using Corel Photo-Paint 12 (CorelDRAW version 12) and UTHSCSA-ImageTool software (version 3.0).

For immunofluorescence staining, the cells were allowed to grow on coverslips and cultured in complete medium at 37°C, 5% CO₂. Cells were fixed with ice-cold methanol for 10 min; then excess methanol was removed. After three subsequent washes with PBS, the cells were blocked with protein blocker (Dako Cytomation, Denmark) and then incubated with the primary antibody mouse anti-PrlR clone 1A2B1 in dilution of 1:50 (Invitrogen) over night at 4°C, washed 3 times with PBS; and then incubated for 1 h at room temperature in the dark with the secondary antibody Alexa fluor 488- conjugated goat anti-mouse IgG (Invitrogen). Finally, the slides were washed, mounted with Vectra shield containing DAPI (H-1200, Vector Laboratories, CA, USA) and stored at 4°C in the dark. Controls for specificity and background staining were performed by incubating samples with mouse IgG antibody. Confocal Laser Scanning Microscopy (CLSM) (Zeiss, Germany) was used to obtain the images.

Sections 5 μm in thickness were de-paraffinised and re-hydrated. A pretreatment method using heat-induced epitope retrieval was used, and the nonspecific binding was blocked with a protein blocker (DakoCytomation, Trappe, France) for 30 min at room temperature (RT). The sections were then incubated with the following primary antibodies against Ki67 (ab15580; Abcam), EpCam (clone G8.8; Biolegend), α-SMA (ab21027; Abcam), CD34 (RAM34, eBioscience), GP38 (MA615113; Thermo Fisher), CD24 (ab64064; Abcam), Lysozyme (ab108508; Abcam), Muc2 (Thermo Fisher) and ZO-1 (Invitrogen) antibodies.
The co-labelling with antibodies from the same species was performed using Opal™ Multiplex IHC kits (Akoya). After incubation with the primary antibodies, the slides were washed in PBS-T three times and probed with appropriated fluorescence-conjugated secondary antibodies for 1 h at room temperature. After three washings in PBS, the cell nuclei were counterstained by Vectashield mounting medium with DAPI (Vector).