Dylight 488

Caki-2 and 786-O cells were allowed to adhere to WillCo-35-mm confocal dishes (WPI, Sarasota, FL, Invitrogen) with a glass bottom. 50 IU/mL r-Hu EPO was added to the dish and cells were incubated for 48 h at 37°C. Cells were washed with PBS and fixed with 4% pre-warmed formaldehyde for 20 to 30 min at room temperature, following by permeabilization with 0.1% Triton X-100. The permeabilized cells were stained with KIAA0101 (diluted 1:500, Abcam, Cambridge, MA, USA) and DyLight 488 (diluted 1:1,000, EarthOx, San Francisco, CA, USA) antibodies. For image acquisition, cellular fluorescence was scanned and monitored with an UltraVIEW VOX Confocal imaging system (PerkinElmer, Cambridge, UK).

With regard to the immunofluorescence method, myocardial tissue slices were incubated with the following primary antibodies: Rabbit anti-NLRP3 (1:100, Proteintech, 19771-1-AP, United States), anti-Caspase 1 (1:50, Proteintech, 22915-1-AP, United States), anti-IL-1β (1:200, Proteintech, 26048-1-AP, United States), anti-IL-18 (1:400, Proteintech, 10663-1-AP, China), anti-ASC (1:800, Cell Signaling, #67824, United States).
The slices were then washed and detected with appropriate Fluor dye, DyLight 488 (1:50, EarthOx, E032220-01, United States), as secondary antibodies followed by counterstaining with DAPI in the dark. Afterward, immunofluorescent signaling was observed with a fluorescence microscope (IX73 Olympus). Digital images and data were recorded and analyzed by applying ImageJ (NIH).

Immunofluorescence staining of intracellular albumin and CYP3A4 was conducted to examine a liver cell-specific function within the BSAMA cryogels. At day 1, day 7, and day 14, cell-laden samples were washed with PBS for three times, and fixed with 4% paraformaldehyde (PFA) for 5 min, and then blocked and permeated with TritonX-100 (0.1% w/v) in a BSA solution (1% w/v in PBS) for 1 h at 4 °C. The samples were incubated with either rabbit anti-albumin primary antibody (1:100, Affinity Biosciences) or mouse anti-CYP3A4 primary antibody (1:100, Affinity Biosciences) in a BSA solution (1% w/v) at 4 °C overnight. After that, these samples were washed with PBS for three times and incubated with either goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody, both conjugated with DyLight 488 (1:50, Earthox), for 2 h at room temperature. Before the images of cross-sections were captured, these samples were split in the middle of the surface vertically. Confocal microscopic images of CYP3A4 and albumin expression were obtained with a confocal laser scanning microscope (Nikon A1, Tokyo, Japan).

Cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 20 min at room temperature. Cells were incubated with primary antibodies overnight at 4 °C after blocking with 10% goat serum. After washing with 1 × PBS, the cells were incubated with secondary antibodies conjugated with DyLight 488 or DyLight 594 (1:100) (Earthox, Millbrae, CA, USA) for 1 h at 37 °C. The cells were stained with DAPI (Beyotime, Nantong, China) to visualize the nuclei.

Differentiated podocytes were fixed with cold methanol at −20°C for 20 min and permeabilized with 0.1% Triton X-100/PBS. After they were washed three times with cold PBS. The cells were blocked with 2% bovine serum albumin (BSA) for 1 h at room temperature and incubated with mouse anti-synaptopodin antibody (1:200, santa cruz biotechnology, sc-515842) or rabbit anti-cleaved caspase three antibody (1:400 Cell Signaling Technology, 9661) at 37°C for 2 h or 4°C overnight. Then, the cells were washed three times with PBS and incubated with a secondary antibody conjugated to DyLight 488 or DyLight 594 (Earthox, Millbrae, CA, United States) at 37°C for 1 h, respectively. Nuclei were counterstained with DAPI. The coverslips were mounted onto glass slides, and the images were viewed with an Olympus BX43F fluorescence microscope (OLYMPUS, Japan).