MA5-14520

antibody from Invitrogen Antibodies

Targeting: MKI67 MIB-1, PPP1R105

Provider product page for MA5-14520

Western blot

Antibody data

Antibody Data
Antigen structure
References [770]
Comments [0]
Validations
Immunocytochemistry [2]
Immunohistochemistry [6]
Flow cytometry [1]
Other assay [4]

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Product number: MA5-14520 - Provider product page
Provider: Invitrogen Antibodies
Product name: Ki-67 Recombinant Rabbit Monoclonal Antibody (SP6)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Antibody clone number: SP6
Vial size: 500 µL
Concentration: 0.031 mg/mL
Storage: Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

MKI67 protein structure - MA5-14520 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of Ki-67 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR955684_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Immunofluorescence analysis was performed on HeLa wild type cells (panel a,d), HeLa Cas9 control cells (panel b,e) and HeLa Ki-67 KO cells (panel c, f). Cells were fixed, permeabilized, and labeled with Ki-67 Recombinant Rabbit Monoclonal Antibody (SP6) (Product # MA5-14520, 1:500 dilution), followed by Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Product#A32790, 1:2000 dilution). Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal (panel c,f) upon CRISPR mediated knockout (KO) confirms that antibody is specific to Ki-67 (green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Ki-67 was performed using 70% confluent log phase HeLa cells serum starved (SS) for 36 hours followed by serum release (SR) for 6 hours. 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 labeled with Ki-67 Monoclonal Antibody (SP6) (Product # MA5-14520) at 1:250 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790) at a dilution of 1:2000 for 45 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents the serum starved cells with reduced signal. 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: Formalin-fixed, paraffin-embedded human high grade invasive breast cancer tissue stained with MA5-14520 (1:400) to detect Ki-67, followed by a peroxidase-conjugated secondary reagent and DAB substrate. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Formalin-fixed, paraffin-embedded human tonsil stained with Ki-67 using peroxidase-conjugate and AEC. Note nuclear staining of proliferating cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Ki-67 (SP6) showing staining in the nucleus of paraffin-embedded human tonsil tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Ki-67 Antibody (SP6) Rabbit Monoclonal Antibody (Product # MA5-14520) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Ki-67 (SP6) showing staining in the nucleus of paraffin-embedded human breast tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Ki-67 Antibody (SP6) Rabbit Monoclonal Antibody (Product # MA5-14520) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical analysis of Ki-67 was performed using formalin-fixed paraffin-embedded human colon adenocarcinoma 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 Rabbit Monoclonal Antibody (SP6) (Product # MA5-14520) at 1:100 dilution in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. Detection was performed using Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical analysis of Ki-67 was performed using formalin-fixed paraffin-embedded human colon adenocarcinoma 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 or without Ki-67 Recombinant Rabbit Monoclonal Antibody (SP6) (Product # MA5-14520) at 1:100 dilution in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. Detection was performed using Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 Col2-Cre: Tsc1 fl/fl metatarsals have a growth advantage ex vivo . ( A ) Growth curves of metatarsals collected from three-day old mice and cultured for six days ex vivo . ( B ) Metatarsals were collected immediately before culture or after one day in culture (contralateral controls), then stained for pS6, Ki67 (immunohistochemistry) or COL10A1 ( in situ hybridization). For Ki67 staining, the region directly above the hypertrophic zone is expanded (pink box). For COL10A1 staining, the yellow dashed line denotes the border between the cartilage and the primary spongiosa. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 2

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 RSPO2 Promotes Midbrain Dopaminergic Neurons Differentiation in Primary Cultures (A) Schematic representation of the mouse primary cell culture protocol. (B) Quantification of the total cell numbers (DAPI) and proportion of neurons (betaIII-tubulin/DAPI) normalized to control (Vehicle). (C) Quantification of ongoing cell death, measured by active caspase-3 ICC (aC3 + /DAPI), showed no difference compared with Vehicle. (D) Quantification of KI67 ICC (KI67 + /DAPI) showed no difference compared with Vehicle. (E) ICC staining showing and increase in TH + neurons compared with DAPI after RSPO2 treatment, but not RSPO1 or SPON1. Scale bars, 50 mum. (F) Quantification of postmitotic cells in the DA lineage, measured by NR4A2 ICC, showed no difference compared with Vehicle. (G) Bar plots showing that RSPO2 treatment increases the total number of TH + cells (TH/DAPI), compared with control (Vehicle). (H) Normalized ratio of TH + cells/NR4A2 + cells revealed an increase in the proportion of postmitotic dopaminergic cells (NR4A2 + ) becoming mDA neurons (TH + ). (I) ICC staining of cells treated with various Wnt signaling pathway modifiers, stained with TH and betaIII-tubulin. Scale bars, 50 mum. (J) Quantification of TH + cells in various treatments from (I) as compared with Vehicle. All data normalized to control (Vehicle) presented as means +- SEM. n = 3, t test; * p < 0.05, ** p < 0.01. See also Figure S3 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 RSPO2 Promotes the Dopaminergic Differentiation of Mouse ESCs (A) Cartoon showing the protocol for the differentiation of mouse ESCs into mDA neurons and the time line (d, days) of spondin treatment, between days 5 and 11. (B) Quantification of proliferation (KI67) in areas undergoing neurogenesis (betaIII-tubulin + ) as assessed by ICC, normalized to control. (C) Quantification of ongoing cell death by active caspase-3 (aC3) ICC in betaIII-tubulin + areas. (D) ICC for NR4A2 and betaIII-tubulin after 14 days in vitro and bar plots showing no difference in the number of NR4A2 + cells per betaIII-tubulin area. Scale bars, 50 mum. (E) ICC staining for TH and betaIII-tubulin after 14 days in vitro in vehicle-, SPON1-, and RSPO2-treated cultures. Scale bars, 50 mum. (F) The number of TH + cells per betaIII-tubulin area by ICC, normalized to control, increased by RSPO2 treatment and decreased by SPON1. (G) The proportion of NR4A2 + cells that become TH + increased by RSPO2 treatment and decreased by SPON1. All data normalized to control (Vehicle) presented as means +- SEM. n = 3, t test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 Decreased parietal cells and increased cell proliferation in Helicobacter -infected Lgr5-Cre;Bmpr1a flox-flox mice. One- to 2-month-old Lgr5-Cre mice and Lgr5-Cre;Bmpr1a flox-flox mice were treated with 1 intraperitoneal injection of tamoxifen (0.1 mg/g body weight) and inoculated with H felis 2 months after tamoxifen. Animals were analyzed 3 months after inoculation and 5 months after tamoxifen injection. Paraffin sections of the lesser curvature of Lgr5-Cre mice and of Lgr5-Cre;Bmpr1a flox-flox mice, in the presence and absence of H felis (H. f.), were stained with an ( A ) anti-H + ,K + -ATPase alpha-subunit primary antibody and an Alexa 488-conjugated secondary antibody (green), and with an ( C ) anti-Ki67 primary antibody and an Alexa 555-conjugated secondary antibody (red). Scale bars : 100 mum. Bars represent the number of ( B ) H + ,K + -ATPase alpha-subunit and of ( D ) Ki67-positive cells detected in Lgr5-Cre mice and in Lgr5-Cre;Bmpr1a flox-flox mice in the presence and absence of H felis . Values are shown as means +- SE. Numbers in parenthesis indicate the number of animals used in each group. * P < .05 vs Lgr5-Cre mice. ** P < .05 vs Lgr5-Cre;Bmpr1a flox-flox mice. DAPI, 4'6-diamidino-2-phenylindole.