MA5-16409

antibody from Invitrogen Antibodies

Targeting: VIM

Provider product page for MA5-16409

Western blot

Immunocytochemistry

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [41]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [2]
Immunohistochemistry [2]
Other assay [7]

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Product number: MA5-16409 - Provider product page
Provider: Invitrogen Antibodies
Product name: Vimentin Monoclonal Antibody (SP20)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: Predicted to react with Rat, Hamster, Cow, and Xenopus laevis based on sequence homology. This antibody is not suitable for mouse tissues. Recommended Postive Controls - WB: U-2 OS, Hu tonsil and HeLa cell lysates. Flow: HeLa cells. ICC/IF: HAP1-VIM cells, human limbal epithelial cells. IHC-P: Human breast cancer and melanoma tissue. IHC-Fr: Colorectal cancer tissue.
Reactivity: Human
Host: Rabbit
Isotype: IgG
Antibody clone number: SP20
Vial size: 500 µL
Concentration: 0.006 mg/mL
Storage: Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

VIM protein structure - MA5-16409 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), SHSY-5Y (Lane 3), HepG2 (Lane 4) and NIH/3T3 (Lane 5). The blot was probed with Anti-Vimentin Monoclonal Antibody (Product # MA5-16409, 1:2000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 54 kDa band corresponding to Vimentin was observed across all the human cell lines positive for Vimentin (Lanes 1 and 3) but not mouse cell line (Lane 5), while this band was absent in the cell lines which do not express Vimentin protein (Lanes 2 and 4).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Vimentin Monoclonal Antibody (SP20) (Product # MA5-16409) and a ~58kDa band corresponding to vimentin was observed across cell lines tested except in A-431. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), SH-SY5Y (Lane 2), A-549 (Lane 3), A-431 (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX), 12 well. Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Hela Cells using anti-Vimentin Monoclonal Antibody (Product # MA5-16409). The recommened dilution for this antibody in western blot applications is 1:100.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of vimentin was performed using 70% confluent log phase 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 labeled with Vimentin Monoclonal Antibody (SP20) (Product # MA5-16409) at 1:1000 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), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Alexa Fluor™ Plus 647 Phalloidin (Product # A30107, 1:2000 dilution). Panel d represents the merged image showing cytoplasmic and cytoskeletal localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification in Cell Insight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Vimentin was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Vimentin Rabbit Monoclonal Antibody (Product # MA5-16409) at 1:200 dilution in 0.1% BSA and incubated overnight at 4 degree Celsius and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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 cytoplasmic, cytoskeletal and nuclear localization. Panel e represents negative control, A-431 cells. 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)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Fabrication of cardiac spheroids for disease modeling applications. a (i) Development of healthy and scarred spheroids through mixing iPSC-derived cardiomyocytes (iPSC-CMs) with primary adult cardiac fibroblasts (CFs) at defined ratios of cell numbers (4:1 for healthy; 1:4 for scarred). Top: Images (cardiac troponin-T (cTnT) (red; iPSC-CMs); vimentin (green; CFs)) taken 3 days after cell seeding. Scalebar 50 um. Bottom: Immunofluorescence staining for alpha-actinin (green; sarcomeres) and cTnT (red; iPSC-CMs) in healthy and scarred spheroids at 3 days. Scalebar 10 um. (ii) Quantification of cellular composition through staining for cTnT (iPSC-CMs) and vimentin (CFs) ( n = 3 biologically independent samples, mean +- s.d, two-sided student t test, healthy - cTnT + vs. Vimentin + p = 5.6 x 10 -5 , scarred - cTnT + vs. Vimentin + p = 6.7 x 10 -4 ). b (i) Contraction profiles, (ii) contraction amplitude (a.u. absolute units), and (iii) peak-to-peak time (ms) of healthy and scarred cardiac spheroids at 3 days ( n = 3 biologically independent samples, mean +- s.d, two-sided student t test, (ii) p = 5.0 x 10 -4 ). c Quantification of calcium activation parameters from calcium mapping experiments in healthy and scarred spheroids at 3 days, including (i) calcium transient duration (ms), (ii) time-to-peak (ms), and (iii) calcium flux amplitude (F/Fo) ( n = 5 biologically independent samples, mean +- s.d, two-sided student t test, (i) p = 0.0014, (ii) p = 5.0 x 10 -6 , (iii) p = 2

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 3D bioprinting cardiac microtissues for disease modeling applications. a (i) Schematic of 3D bioprinting of healthy and scarred cardiac microtissue rings and (ii) immunofluorescence staining for cTnT and vimentin in healthy and scarred cardiac microtissues after 5 days of fusion within the support hydrogel. Scalebar 100 um (insets 50 um). (iii) Immunofluorescence staining for alpha-actinin (green; sarcomeres) and cTnT (red; iPSC-CMs) and (iv) connexin-43 (green; gap junctions) and cTnT (red; iPSC-CMs), in healthy and scarred regions of microtissues after 5 days of fusion within the support hydrogel. Scalebar 10 um. b (i) Contraction profiles of healthy and scarred cardiac microtissues following removal from the support hydrogel after 5 days of culture. (ii) Contraction amplitude (a.u. absolute units) and (iii) Peak-to-peak time (ms) at 5 days ( n = 3 biologically independent samples, mean +- s.d, two-sided student t test, (ii) p = 0.061). c (i) Calcium mapping in healthy and scarred cardiac microtissues after 5 days culture, each image represents a 20 ms frame. Scalebar 100 um. (ii) Representative calcium traces from regions 1 and 2 (see methods) in healthy and scarred cardiac microtissues. Scalebars 0.5 DeltaF/F o (y), 500 ms (x). (iii) Activation maps of healthy and scarred cardiac microtissues, and activation delay (ms) (difference in activation time (ms) between regions 1 and 2) in healthy and scarred cardiac microtissues ( n = 3 biologically independent samples, m

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6 Evaluating miRNA on the behavior of cardiac microtissues. a (i) Schematic of cholesterol modified miR302 (chol-miRNA 302 b/c) delivery to scarred cardiac spheroids for 0, 0-2, 0-4, and 0-7 days. (ii) Contraction amplitude (a.u) and (iii) peak-to-peak time (ms) within scarred spheroids measured after 2, 4, and 7 days for each treatment period ( n = 4, 6, 5, 5, 7, 5, 5, 5, 5 biologically independent samples (from left to right), mean +- s.d, one-way ANOVA, (ii) day 4: 0 vs. 0-4 days treatment p = 0.014, (ii) day 7: 0 vs. 0-7 days treatment p = 0.0045, (iii) day 4: 0 vs. 0-4 days treatment p = 1.6 x 10 -7 , (iii) day 7: 0 vs. 0-7 days treatment p = 4.1 x 10 -9 ). b (i) Immunofluorescence staining for cTnT (red; iPSC-CMs), vimentin (green; cardiac fibroblasts), and EdU (proliferation marker) in scarred spheroids at day 7 for each treatment condition. Top panel scalebar 50 um, and bottom panel scalebar 40 um. (ii) Quantification of cardiomyocyte proliferation (EdU + and cTnT + ) and (iii) fibroblast proliferation (EdU + and Vimentin + ) at day 7 for each treatment condition. ( n = 3, 4, 4, 4 biologically independent samples (from left to right), mean +- s.d, one-way ANOVA, 0 vs. 0-4 days treatment p = 0.044, 0 vs. 0-7 days treatment p = 0.026). Scalebar 50 um. c (i) Experimental outline where scarred microtissues are bioprinted in the support hydrogel as previously described, followed by 4 days treatment with miR302, and analysis compared to non-treated controls. (ii) Calci

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 The effect of MMP-9 downregulation on vimentin, N-cadherin and F-actin level. NT--non-transfected cells, T--cells transfected with MMP-9 siRNA, NT5 and T5 non-transfected and transfected cells treated with 5 mM cyclophosphamide for 24 h. ( A ) Immunofluorescent imaging of vimentin (red) and nuclei (blue), and relative fluorescence intensity of vimentin. ( B ) Immunofluorescent imaging of N-cadherin (red) and nuclei (blue), and relative fluorescence intensity of N-cadherin. ( C ), Immunofluorescent imaging of F-actin (green), and nuclei (blue), Bar = 50 mum. ( D ) The Western blot analysis of vimentin and N-cadherin and densitometric relative expression level of the Western blot experiments. Statistically significant differences were marked with '*' p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 ( A , B ) Cell EMT-related protein levels of E-cadherin, N-cadherin, Vimentin and beta-actin in AGS and ( C , D ) HGC-27 cells were investigated by Western blot. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 2 Pi3K pathway plays a major role in mesenchymal phenotype. (A) The correlation of EMT score with protein expression data of TCGA patients was calculated and plotted. The Yellow dots represent the positive correlation (>+0.2), and the green dots represent the negative correlation (