Fluorescein fitc conjugated affinipure goat anti mouse igg h l

For immunofluorescence staining, tissue sections were fixed with 4% paraformaldehyde for 20 min, incubated with permeabilization solution (0.1% Triton × 100 and 0.1% sodium citrate) on ice for 2 min and blocked with horse serum for 2 h. Sections were stained with antibodies against Mac3 (1:200, Cat^# ab199947, Abcam), CD31 (1:200, Cat^# sc-376764, Santa Cruz Biotechnology), α-SMA (1:200, Cat^#sc-53142, Santa Cruz Biotechnology), or AIBP (1:200, Cat^# ab75114, Abcam) followed by detection with fluorochrome-conjugated secondary antibodies (fluorescein (FITC)–conjugated Affinipure goat anti-rabbit IgG (H + L), Cat^# SA00003-2, Proteintech; fluorescein (FITC)–conjugated Affinipure goat anti-mouse IgG (H + L), Cat^# SA00003-1, Proteintech; Cy3–conjugated Affinipure goat anti-rabbit IgG (H + L), Cat^# SA00009-2, Proteintech; and Cy3–conjugated Affinipure goat anti-mouse IgG (H + L), Cat^# SA00009-1, Proteintech). Sections were counterstained with DAPI (Cat# BS097, Biosharp), coverslipped and then scanned with a fluorescence microscope (IX70; Olympus, Tokyo). Negative controls were obtained by incubating tissue sections with the corresponding secondary antibodies alone. Images were analyzed using ImageJ 2X software (Media Cybernetics, Bethesda, MD).

The expression of the ALDH1A1 marker was evaluated using flow cytometry, adhering strictly to the manufacturer’s protocol. We utilized the primary anti‐ALDH1A1 antibody at specific working dilutions (0.2 μg of 60171-1-Ig, Clone:1A10A2, sourced from Proteintech, USA). This was followed by the application of the secondary antibody, Fluorescein (FITC)–conjugated Affinipure Goat Anti-Mouse IgG(H + L), at a dilution ranging between 1:20 and 1:100 (also provided by Proteintech, USA).

Renal cell carcinoma cell lines 786O, OSRC2, 769P, ACHN, CAKI2, and normal cell HK2 were purchased from ATCC. HK2 cells were cultured in DMEM/F12 medium (Gibco, USA) containing 10% fetal bovine serum (Excell, China), renal cell carcinoma cells 786O, OSRC2, 769P, and ACHN were cultured in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum, and CAKI2 cells were cultured in McCoy’s 5A medium (Procell, China) containing 10% fetal bovine serum. All cell lines were incubated at 37 ° with 5% CO₂.
Primary antibody against pan Keratin was bought from Cell Signaling Technology (Danvers, USA), Fluorescein (FITC)–conjugated Affinipure Goat Anti-Mouse IgG(H + L) was bought from Proteintech Group Inc. (USA).