Rhodamine labeled wheat germ agglutinin

The presence of human stromal cells in the kidney, lung, liver, and heart of RMR rats was determined as previously described^{17 (link)}
. Tissue sections were incubated for 1 hour with a 1% bovine serum albumin blocking solution, followed by incubation with Alexa Fluor 488 conjugated anti-human nuclear antigen (HNA) antibody (1:50; clone 235 -1, Merck Millipore) overnight at 4 °C. Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Merck Millipore), and the tissue structures were marked using Rhodamine-labeled wheat germ agglutinin (WGA, 1:400; Vector Laboratories, Burlingame, CA, USA).

Cells were seeded (1.2 × 10⁴ cells/well) on 8-well chamber slides (Falcon™, Corning Brand, USA) in growth medium with 10% FCS and allowed to recover for 48 h. To determine the cellular uptake of albumin, the cells were treated with 10 µM FITC-labeled albumin (dissolved in serum-free RPMI medium) for 3 h and subsequently fixated using a solution of 4% paraformaldehyde dissolved in PBS with the pH adjusted to 7.4 (Merck, Darmstadt, Germany) for 15 min at room temperature. Spots were washed 4-timeswith PBS. Additionally, a staining solution of 0.3% DAPI and 0.45% rhodamine-labeled wheat germ agglutinin (WGA) (Vector laboratories, Newark, CA, USA) was added to stain cell nuclei and cell membranes, respectively. Next, the slide was mounted with non-hardening mounting medium (Vectashield^® Mounting Media, Vector laboratories, Newark, CA, USA), and analyzed using fluorescence measurement with a confocal microscope (Zeiss, Oberkochen, Germany) and image processing using ZEN lite software (Zeiss, Oberkochen, Germany).