RT KI67 PU RMAB

Immunofluorescent analysis of Ki-67 was performed using 70% confluent HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes and blocked with 2% BSA for 1 hour at room temperature. The cells were stained with Ki-67 Recombinant Rat Monoclonal Antibody (SolA15) (Product # 740008T, 1:400) at 4 degree Celsius overnight and then labeled with Donkey Anti-Rat Secondary Antibody, Alexa Fluor™ 647 (Product # A78947) at a dilution of 1:2,000 for 1 hour at room temperature. Panel a) shows representative images of cells that were stained for detection and localization of Ki-67. Panel b) shows representative images of cells stained for nuclei using SYTOX™ Green Nucleic Acid Stain (Product # S7020, 1:10,000). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor™ Plus 405 Phalloidin (Product # A30104, 1:300). The slides were mounted using ProLong™ Diamond Antifade Mountant (Product # P36961). Panel d) is a composite image of panels a, b, and c, clearly demonstrating nuclear localization of Ki-67. Panel e) 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, AssayID CRISPR955686_LV) and LentiArray Cas9 Lentivirus (Product # A32069). Immunofluorescence analysis was performed on wild type HeLa cells (panel a-d), HeLa Cas9 control cells (panel e-h) and HeLa Ki-67 KO cells (panel i-l). Cells were fixed, permeabilized, and labelled with Ki-67 Recombinant Rat Monoclonal Antibody (SolA15) (Product # 740008T, 1:400), at 4 degree Celsius overnight and then labeled with Donkey Anti-Rat Secondary Antibody, Alexa Fluor™ 647 (Product # A78947) at a dilution of 1:2,000 for 1 hour at room temperature. Nuclei (green) were stained using SYTOX™ Green Nucleic Acid Stain (Product # S7020, 1:10,000) and Alexa Fluor™ Plus 405 Phalloidin (Product # A30104, 1:300) was used for cytoskeletal F-actin (blue) staining. The slides were mounted using ProLong™ Diamond Antifade Mountant (Product # P36961). Loss of signal (panel i,l) upon CRISPR mediated knockout (KO) confirms that the antibody is specific to Ki-67 (red). The images were captured at 60X magnification.&(8)203;

Immunofluorescent analysis of Ki-67 was performed using 70% confluent HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes and blocked with 2% BSA for 1 hour at room temperature. The cells were stained with Ki-67 Recombinant Rat Monoclonal Antibody (SolA15) (Product # 740008T, 1:1,000) at 4 degree Celsius overnight and then labeled with Donkey Anti-Rat Secondary Antibody, Alexa Fluor™ 647 (Product # A78947) at a dilution of 1:2,000 for 1 hour at room temperature. Panel a) shows representative images of cells that were stained for detection and localization of Ki-67. Panel b) shows representative images of cells stained for nuclei using SYTOX™ Green Nucleic Acid Stain (Product # S7020, 1:10,000). 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 20X magnification using CellInsight™ CX7 LZR High-Content Screening (HCS) Platform.

Immunofluorescence analysis of Ki-67 was performed using 70% confluent HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes and blocked with 2% BSA for 45 minutes at room temperature. The cells were labelled with Ki-67 Recombinant Rat Monoclonal Antibody (SolA15) (Product # 740008T, 1,10,000l) in 0.1% BSA, incubated at 4 degree Celsius overnight and then labelled with Donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A48269, 1:2,000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents HeLa cells showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification.

Immunohistochemical analysis of Ki-67 was performed using formalin-fixed paraffin-embedded mouse thymus tissue sections. To expose the target protein, heat-induced epitope retrieval was performed on de-paraffinized sections using eBioscience™ IHC Antigen Retrieval Solution - High pH (10X) (Product # 00-4956-58) 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 and then probed with Ki-67 Recombinant Rat Monoclonal Antibody (SolA15) (Product # 740008T) at 0.5 µg/mL in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. Detection was performed using Goat anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 594 (Product # A48264, 1:2,000) in 0.1% normal goat serum for 45 minutes at room temperature. 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 and externally deconvoluted. The antibody stains specifically to the nucleus of cells in the thymus, as shown in the figure.

Immunohistochemical analysis of Ki-67 was performed using formalin-fixed paraffin-embedded mouse thymus 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 and then probed with Ki-67 Recombinant Rat Monoclonal Antibody (SolA15) (Product # 740008T) at 0.5 µg/mL in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. Detection was performed using Goat anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 594 (Product # A48264, 1:2,000) in 0.1% normal goat serum for 45 minutes at room temperature. 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 and externally deconvoluted. The antibody stains specifically to the nucleus of cells in the thymus, as shown in the figure.

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 - High pH (10X) (Product # 00-4956-58) 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 and then probed with or without Ki-67 Recombinant Rat Monoclonal Antibody (Product # 740008T) at 1:200 dilution in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. Detection was performed using Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (Product # A11006) at a dilution of 1:2,000 in 0.1% normal goat serum for 1 hour at room temperature. 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 and externally deconvoluted.

HeLa cells were fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (Product # 00-5523-00) and then stained intracellularly with 0.063 µg of Ki-67 Recombinant Rat Monoclonal Antibody (SolA15) (Product # 740008TM) (pink histogram) or 0.063 µg of Rat IgG2a kappa Isotype Control (Product # 14-4321-85) (blue histogram), followed by Donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (Product # A78947). 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 target protein in Ki-67 KO cell line compared to control cell line, using Ki-67 Recombinant Rat Monoclonal Antibody (SolA15) (Product # 740008T). {KO}