- 701198 - Invitrogen Antibodies MKI67 antibody

Hu W, Liu Z, Salato V, North PE, Bischoff J, Kumar SN, Fang Z, Rajan S, Hussain MM, Miao QR
JCI insight 2021 Feb 8;6(3)

Dong Q, Pan S, Sun LS, Li TJ
Archives of pathology & laboratory medicine 2010 Feb;134(2):271-5

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Ki-67 was performed on HeLa cells serum-starved for about 16 hours and then serum released for 4 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0. 25% of Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Ki-67 Recombinant Rabbit Monoclonal Antibody (Product # 701198) at a dilution of 1:400 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG secondary antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 phalloidin (Product # A12381). Panel d is a merged image showing nuclear localization. The images were captured using a Nikon microscope at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Proliferation marker protein Ki-67 was performed using 70% confluent log phase HeLa cells (Serum release,8 Hrs). 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 labeled with Ki-67 Recombinant Rabbit Monoclonal Antibody (12H15 L5) (Product # 701198) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000 dilution), 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 dilution). Panel d represents the merged image showing nuclear localization. Panel e represents HeLa (serum starved for 16Hrs). Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of SOX2 (green) and Ki67 (red) in human iPSC-derived forebrain organoids derived at Day 40. The organoids were fixed with 4% PFA for 1 hour at room temperature, followed by incubation with 30% sucrose solution overnight at 4°C. The organoids were then embedded in OCT and cryosectioned at 5 µm, permeabilized with 0.2% Triton X-100 for 20 min, and blocked with 10% donkey serum in PBS for 30 min at room temperature. Organoid slices were stained with a Mouse SOX2 monoclonal antibody (green; Product # MA1-014) at a dilution of 1:500, and a Rabbit Ki67 monoclonal antibody (red; Product # 701198) at a dilution of 1:500 in blocking buffer overnight at 4°C, and then incubated with Donkey anti-Mouse Alexa Fluor 488 (Product # R37114) and Donkey anti-Rabbit Alexa Fluor 568 (Product # A10042) at a dilution of 1:1000 as well as DAPI (blue; 1:25000) in blocking solution at room temperature for 1 hour. Images were taken at 20X magnification. Data courtesy of Dr. Zhexing Wen at Emory University.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of Ki-67 was performed on serum-starved HeLa cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0. 25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with ABfinityª Ki-67 recombinant rabbit monoclonal antibody (Product # 701198, red histogram) or with rabbit isotype control (pink histogram) at a dilution of 1:400 in 2.5% BSA. After incubation at room temperature for 3 hours, the cells were labeled with Alexa Fluor¨ 488 goat anti-Rabbit Secondary antibody (Product # A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Chromatin immunoprecipitation analysis of Ki-67 was performed using cross-linked chromatin from 1 x 10^6 HCT116 human colon carcinoma cells treated with serum for 0, 15, and 30 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of a Ki-67 rabbit monoclonal antibody (Product # 701198). Chromatin aliquots from ~1 x 10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify-15kb upstream of the human Egr-1 locus, or exon-1 or exon-2 of Egr-1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. Data courtesy of the Innovators Program.

701198