4 6 diaminido 2 phenylindol dapi

At 25 days after the operation, the ischemic thigh tissues were removed and fixed with 4% paraformaldehyde (Sigma), and each tissue sample was embedded in paraffin. For histological analysis, the samples were stained with H&E, or Masson's trichrome in kidney tissues to determine fibrosis and histopathological features, respectively. Immunofluorescence staining was performed with primary antibodies against CD11b (abcam), CD31 (Santa Cruz Biotechnology), and α-SMA (alpha-smooth muscle actin; Santa Cruz Biotechnology), followed by secondary antibodies conjugated with Alexa Fluor 488 or 594 (Thermo Fisher Scientific). Nuclei were stained with 4′,6-diaminido-2-phenylindol (DAPI; Sigma), and the immunostained samples were examined by confocal microscopy (Olympus, Tokyo, Japan).

Cortical kidney tissue samples were collected, immediately fixed with 4%
paraformaldehyde (CureBio, Seoul, Korea), and then embedded in paraffin. For
histological analysis, the kidney tissue samples were stained with Masson’s
trichrome (MT) or Picro Sirius red (PSR) to determine the presence of fibrosis
and the stained tissues were examined using light microscopy (U-TVO 63XC;
Olympus Corp., Tokyo, Japan). The MT and PSR stained area were measured using
Image J software with installed NII plugin (National Institutes of Health, MD, USA).^{4 (link)}
Immunofluorescence staining was also performed with primary antibodies against
cleaved caspase 3 (Novus Biologicals, Denver, CO, USA), proliferating cell
nuclear antigen (PCNA, Abcam), and α-smooth muscle actin (SMA; Santa Cruz
Biotechnology), followed by secondary antibodies conjugated with Alexa Fluor 488
or 594 (Thermo Fisher Scientific). The nuclei were stained with
4,6-diaminido-2-phenylindol (DAPI, Sigma-Aldrich), and the immunostained samples
were examined using a confocal microscope (Olympus, Tokyo, Japan). The apoptosis
(or proliferation) index was determined as number of caspase-3 (or PCNA)
positive cells/total number of cells × 100.^{26 (link)}