Western blotting analysis using CD99 antibody (Cat#61390). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with CD99 antibody (Cat#61390, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using FeQ™ ECL Substrate Kit (Cat#226).
Western blotting analysis using CD99 antibody (Cat#61390). CD99 expression in wild type (WT) and CD99 shRNA knockdown (KD) HeLa cells with 30 μg of total cell lysates. Hsp90 α serves as a loading control. The blot was incubated with CD99 antibody (Cat#61390, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using NaQ™ ECL Substrate Kit (Cat#716).
Immunohistochemistry was performed on paraffin-embedded human prostatic adenocarcinoma using CD99 antibody (Cat#61390, 1:200). Antigen retrieval was done in sodium citrate buffer (pH 6.0). DAB was used for detection, with hematoxylin counterstaining. Images were acquired using a Nikon Ci-L Plus microscope (40× objective). Scale bar: 25 μm.
Immunocytochemical staining of HeLa cells using CD99 antibody (Cat#61390, 1:1,000), Top panel: wild-type (WT); Bottom panal: CD99 shRNA knockdown (KD). Nuclei were stained blue with DAPI; CD99 was stained magenta with Alexa Fluor® 647. Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 0.5% Triton X-100 in 1x PBS. Scale bar, 20 μm.
Flow cytometric analysis of CD99 expression in HepG2 cells using CD99 antibody (Cat#61390, 1:2,000). Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol, chill on -20℃ before use. Green, isotype control; red, CD99.
Validation of CD99 knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HeLa cells were stained with CD99 antibody (Cat#61390, 1:2,000) and analyzed using BD flow cytometer. Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol, chill on -20℃ before use.
