RT KI67 AFP647 RMAB

Immunofluorescence analysis of Ki-67 was performed using 70% confluent HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were stained with Ki-67 Recombinant Rat Monoclonal Antibody (SolA15), Alexa Fluor™ Plus 647 (Product # 740008TP647,1:200) at 4 degree Celsius overnight. Panel a) shows representative images of cells that were stained for detection and localization of Ki-67. Panel b) represents cytoskeletal F-actin staining using Alexa Fluor™ Plus 405 Phalloidin (Product # A30104, 1:300). Panel c) is a composite image of panels a, and b, clearly demonstrating nuclear localization of Ki-67. Panel d) represents no primary antibody control to assess the background. The images were captured at 60X magnification.

Knockout of Ki-67 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR955686_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Immunofluorescence analysis was performed on HeLa wild type cells (panel a-c), HeLa Cas9 control cells (panel d-f) and HeLa Ki-67 KO cells (panel g-i). Cells were fixed, permeabilized, and labeled with Ki-67 Recombinant Rat Monoclonal Antibody (SolA15), Alexa Fluor™ Plus 647 (Product # 740008TP647, 1:200 dilution), at 4 degree celsius overnight. Alexa Fluor™ Plus 405 Phalloidin (Product # A30104, 1:300) was used for cytoskeletal F-actin (blue) staining. Loss of signal (panel g-i) upon CRISPR mediated knockout (KO) confirms that antibody is specific to Ki-67 (red). The images were captured at 60X magnification.

Immunohistochemical analysis of Ki-67 was performed using formalin-fixed paraffin-embedded mouse spleen tissue sections. To expose the target protein, heat-induced epitope retrieval was performed on de-paraffinized sections using eBioscience™ IHC Antigen Retrieval Solution - Low pH (10X) (Product # 00-4955-58) diluted to 1X solution in water in a decloaking chamber at 110 degree Celsius for 15 minutes. Following antigen retrieval, the sections were blocked with 2% normal goat serum in 1X PBS for 45 minutes at room temperature, followed by 10 minutes of blocking using Endogenous Biotin-Blocking Kit (Product # E21390) along with 1X PBS wash for 5 minutes. Sections were then probed with or without Ki-67 Recombinant Rat Monoclonal Antibody (SolA15), Alexa Fluor™ Plus 647 (Product # 740008TP647) at 1:100 dilution in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. ReadyProbes™ Tissue Autofluorescence Quenching Kit (Product # R37630) was used to quench autofluorescence from the tissues. Nuclei were stained with DAPI (Product # D1306) and the sections were mounted using ProLong™ Glass Antifade Mountant (Product # P36984). The images were captured on EVOS™ M7000 Imaging System (Product # AMF7000) at 20X magnification.

HeLa cells were fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (Product # 00-5523-00) and then stained intracellularly with 0.25 µg of Ki-67 Recombinant Rat Monoclonal Antibody (SolA15), Alexa Fluor™ Plus 647 (Product # 740008TP647) (pink histogram) or left unstained (blue histogram). Viable cells were used for analysis, as determined by Fixable Viability Dye eFluor™ 450 (Product # 65-0863-18). The flow cytometry data was acquired using Attune™ NxT Flow Cytometer (Product # A29004).

Antibody specificity was demonstrated by CRISPR-Cas9 mediated knockout of target protein. A loss of signal was observed for Ki-67 in KO cell line compared to control cell line using Ki-67 Recombinant Rat Monoclonal Antibody (SolA15), Alexa Fluor™ Plus 647 (740008TP647). {KO}