Cy3 labeled goat anti rabbit igg secondary antibody

Cells in 12-well culture plates were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 (Union Carbide Chemicals and Plastics) in PBS for 20 min at room temperature. Nonspecific antibodies were blocked with 5% bovine serum albumin in PBS for 30 min at room temperature. NTR1 antibody (1:100 dilution) was added to each well and incubated at 4°C overnight to detect NTR1. The sections were then incubated with CY3-labeled goat antirabbit IgG secondary antibody (catalog number GB21303, 1:300 dilution; Servicebio) followed by antifade medium containing 4,6-diamidino-2-phenylindole and were observed under a fluorescence microscope.

The paraffin-embedded sections were dewaxed, and antigen retrieval was performed using EDTA antigen retrieval buffer (Servicebio), followed by three 5-min washes with PBS (7.4). The sections were placed in a 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 min, followed by three PBS washes, for 5 min each. The sections were blocked with 5% BSA (Thermo Fisher Scientific) for 1 h. After removing the blocking solution, an anti-KI67 antibody (1:200; Servicebio) was dropped onto the surface of the section, and the glass slide was placed in a humidified box and incubated overnight at 4°C. The next day, the glass slide was washed three times with PBS, for 5 min each, and then incubated in the dark with a Cy3-labeled goat anti-rabbit IgG secondary antibody (1:1,000; Servicebio) for 2 h. Subsequently, 1 µg/ml DAPI solution was added and the section was incubated for 10 min, followed by three PBS washes, for 5 min each. The stained slides were analyzed under a fluorescence microscope. For DAPI, an excitation wavelength of 330 to 380 nm, and an emission wavelength of 420 nm were used, whereas for Cy3, an excitation wavelength of 510 to 560 nm and an emission wavelength of 590 nm were used.