Goat anti rabbit igg h l alexa fluor 488 secondary antibody

Spheroids were fixed overnight in 4% paraformaldehyde and permeabilized for 5 minutes with 0.1% Triton X-100 in PBS. Samples were then blocked for 1 hour using 0.01% Tween-20, 10% goat serum and 1% bovine serum albumin (BSA) in PBS. Afterwards, samples were incubated with HIF1 alpha primary antibody (Abcam; ab179483; rabbit monoclonal; 1:50) or MMP9 primary antibody (Abcam; ab76003; rabbit monoclonal; 1:250) overnight at 4 degrees, and then incubated with Goat Anti-Rabbit IgG H&L Alexa Fluor® 488 secondary antibody (Abcam; ab150077; goat polyclonal; 2 µg/mL) for 2 hours, as necessary. For F-actin staining, samples were incubated with CytoPainter Phalloidin-iFluor 555 reagent (Abcam; ab176756; 1:1000) for 2 hours. Samples were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Santa Cruz; 1.5 µg/mL). All blocking and incubations were performed at room temperature, unless otherwise stated. Spheroids were visualized using an Eclipse Ti-E Inverted Confocal Microscope (Nikon). Z-stack images were taken in 10 µm steps to capture all layers and compiled as a z-projection image. Images were analyzed using NIS Elements software (Nikon).

Xenograft tumor tissues from mice were paraffin-embedded and sliced at 4 μm. Tumor sections were dewaxed with xylene for 20 min, followed by graded concentrations of ethanol (100%, 95%, 90%, 80%, and 70%) for 5 min at 25 °C. Antigens were obtained by boiling in sodium citrate solution (pH 6.0) for 2 min, and endogenous peroxidase was blocked by soaking in 3% H₂O₂ for 20 min at 25 °C. The sections were incubated with 50 μL of normal goat serum for 15 min at 25 °C without washing. Thereafter, 50 μL of anti-Ki67 (1:200, #ab15580; Abcam, Cambridge, UK) was added to the sections, which were then incubated at 4 °C overnight and washed three times with PBS. Subsequently, the sections were incubated with a goat anti-rabbit IgG H&L (Alexa Fluor® 488) secondary antibody (1:10,000, #ab150077; Abcam, Cambridge, UK) for 15 min at 25 °C. After development with 3, 3′-diaminobenzidine (DAB; Beyotime, Shanghai, China) at 25 °C, the sections were observed under a microscope (CKX53; Olympus, Tokyo, Japan). The sections were then counterstained with hematoxylin for 30 min, rinsed with running water for 10 min, and dehydrated with xylene and ethanol. Finally, images were captured using the microscope.

PK-15 cells were fixed in 4% paraformaldehyde for 30 min at room temperature, washed with PBS three times, permeabilized in 0.25% Triton X-100 for 15 min, and blocked with normal goat serum for 30 min at room temperature. The cells were then incubated with mouse monoclonal antibody (mAb) WH303 specific to CSFV E2 (Bioss, China) overnight at 4 °C, followed by incubation with goat anti-rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody (Abcam, Boston, MA, USA) for 2 h at 37 °C, and washed with PBS three times. Finally, cell nuclei were stained with Hoechst 33,342 (Dojindo, Shanghai, China) for 10 min and observed under a fluorescence microscope.