Cells were fixed with 4% paraformaldehyde for 15 min at 4°C. After washed, the cells were incubated in UltraCruz Blocking Reagent (sc‐516,214; Santa Cruz) supplemented with 0.1% Triton × −100 for 30 min at room temperature. Then, the cells were incubated with anti‐YAP1 antibody (sc‐376,830; Santa Cruz) overnight at 4°C. After 4´,6‐diamidino‐2‐phenylindole staining, YAP expression was observed by EVOS FL Cell Imaging System (Thermo Fisher Scientific).
Anti yap1 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-YAP1 antibody is a laboratory reagent used to detect the presence and quantify the levels of the YAP1 protein in biological samples. YAP1 is a transcriptional co-activator that plays a key role in the Hippo signaling pathway, which regulates cell growth and proliferation. This antibody can be used in various experimental techniques, such as western blotting, immunohistochemistry, and immunoprecipitation, to study the expression and localization of YAP1 in cells and tissues.
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Lab products found in correlation
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Ultracruz blocking reagent, by Santa Cruz Biotechnology (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Chicken anti mouse igg h l alexafluor488, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal laser scanning system, by Olympus (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Axiocam 506 mono camera, by Zeiss (1 mentions)
Anti 53bp1 antibody, by Cell Signaling Technology (1 mentions)
4 protocols using anti yap1 antibody
1
Immunofluorescent Analysis of YAP Expression
2
Immunofluorescence Staining of YAP1 in Cultured Cells
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MED (MIST) 85 and MED (MIST) 96 cells grown at low densities on sterile glass coverslips were fixed in 4% paraform aldehyde for 10 min, permeabilized with 0.4% Triton-X100 in PBS for 15 min, and blocked in 5% BSA for 30 min. Primary antibody staining was performed with anti-YAP1 antibody (Santa Cruz Biotechnology), for 1 h at room temperature, followed by secondary staining with chicken anti-mouse IgG (H+L)-AlexaFluor488 (Thermo Fisher Scientific, 1:1000) for 30 min. Cells were counterstained with DAPI and mounted with ProLong Gold anti-fade reagent (Thermo Fisher Scientific). Confocal imaging was performed using an Olympus FV1000 confocal laser scanning system with an inverted IX81 motorised microscope equipped with UPlanSApo 60×/1.35NA objective (Olympus). Obtained images were deconvoluted using Huygens Essential software (Scientific Volume Imaging) and processed using ImageJ software (NIH).
3
Multimodal Immunofluorescence for DNA Damage
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For YAP1 immunofluorescence staining coupled with EdU incorporation assay, cells were pulse-labeled by 10 μM EdU for 30 min and then fixed by 2% PFA for 15 min at room temperature. Cells were then permeabilized by 0.5% Triton X-100 for 10 min on ice. After washing with PBS, click reaction was performed with freshly prepared click cocktail (2 mM copper sulfate, 10 μM AF488-Azide, and 100 mM sodium ascorbate in PBS) for 1 hr at room temperature. Cells were then preceded to blocking step and YAP1 was stained by anti-YAP1 antibody (Santa Cruz) and Goat anti-Mouse AF568 antibody.
For immunostaining of DNA-damage markers, cells were pre-extracted by 0.5% Triton X-100 in cytoskeletal (CSK) buffer for 3 min on ice and then fixed by 2% PFA for 15 min at room temperature. Total RPA32 was stained by anti-RPA32 antibody (Cell signaling) and Goat anti-Rat TexasRed antibody. 53BP1 was stained by anti-53BP1 antibody (Cell signaling) and Goat anti-Rabbit Dylight 488 antibody. Images were taken using Axioimager.Z1 microscope (Zeiss) equipped with Zeiss AxioCam 506 Mono camera.
For immunostaining of DNA-damage markers, cells were pre-extracted by 0.5% Triton X-100 in cytoskeletal (CSK) buffer for 3 min on ice and then fixed by 2% PFA for 15 min at room temperature. Total RPA32 was stained by anti-RPA32 antibody (Cell signaling) and Goat anti-Rat TexasRed antibody. 53BP1 was stained by anti-53BP1 antibody (Cell signaling) and Goat anti-Rabbit Dylight 488 antibody. Images were taken using Axioimager.Z1 microscope (Zeiss) equipped with Zeiss AxioCam 506 Mono camera.
4
Immunohistochemical Analysis of YAP1 in Breast Cancer
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Hematoxylin and eosin-stained slides from resected breast cancer specimens were examined, and representative areas were marked, and matched tissue cores (2 mm) were extracted from FFPE tumor blocks and placed into 5 × 10 recipient TMA blocks.
For IHC, each TMA slide was stained with an anti-YAP1 antibody (Santa Cruz Biotechnology, Dallas, Texas, USA, 1:200). After staining, nuclear YAP1 expression was assessed by a pathologist using a microscope (400 × magni cation). YAP1 expression was evaluated in both the cytoplasm and nuclei of tumor cells. Cytoplasmic staining was evaluated by the H-score, which was obtained by multiplying staining intensity (0, 1, 2 or 3) by percentage of stained area (%). As nuclear expression was rare and mostly focal, only the intensity of nuclear staining was examined (0, 1+, 2+, 3+), regardless of the corresponding cytoplasmic staining. Intensity of nuclear staining of myoepithelial cells was used as an internal control, and assigned to 2 + value. Weaker and stronger signals were assigned a value of 1 + and 3+, respectively. Negative and weak (1+) nuclear staining were considered indicative of low expression, while moderate (2+) and strong (3+) nuclear expression were indicative of high expression (Fig. 3). The IHC results were interpreted blindly, without any information regarding clinical parameters or outcomes.
For IHC, each TMA slide was stained with an anti-YAP1 antibody (Santa Cruz Biotechnology, Dallas, Texas, USA, 1:200). After staining, nuclear YAP1 expression was assessed by a pathologist using a microscope (400 × magni cation). YAP1 expression was evaluated in both the cytoplasm and nuclei of tumor cells. Cytoplasmic staining was evaluated by the H-score, which was obtained by multiplying staining intensity (0, 1, 2 or 3) by percentage of stained area (%). As nuclear expression was rare and mostly focal, only the intensity of nuclear staining was examined (0, 1+, 2+, 3+), regardless of the corresponding cytoplasmic staining. Intensity of nuclear staining of myoepithelial cells was used as an internal control, and assigned to 2 + value. Weaker and stronger signals were assigned a value of 1 + and 3+, respectively. Negative and weak (1+) nuclear staining were considered indicative of low expression, while moderate (2+) and strong (3+) nuclear expression were indicative of high expression (Fig. 3). The IHC results were interpreted blindly, without any information regarding clinical parameters or outcomes.
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