Cells were fixed using 4% paraformaldehyde/PBS for 15 min at room temperature, washed three times with washing buffer (0.1% Tween-20/PBS, 5 min), and permeabilized with 1% Triton X-100/PBS for 30 min. Samples were incubated in blocking buffer (3% BSA, 2% goat serum in PBS) for 30 min, then incubated with various primary antibodies including mouse anti-YAP (Santa Cruz Biotechnology, sc-101199), mouse anti-αVβ3-integrin (Abcam ab78614), mouse anti-β1-integrin (Abcam ab24693), rabbit anti-paxillin (Abcam ab32084), rabbit anti-pFAK (Abcam ab81298), mouse anti-RUNX2 (Abcam ab76956) overnight at 4 °C on a shaker. All antibodies were diluted in buffer at 1:100 except YAP antibody was diluted at 1:300. After washing, samples were incubated with corresponding secondary antibodies including Alexa 488 Goat-anti-mouse (Invitrogen A11001), rhodamine goat-anti-rabbit (Millipore AP132), rhodamine-phalloidin (Sigma P1951) for 1 h at room temperature on a shaker. All secondary antibodies were diluted at 1:300. Cell nucleus counter stain was performed using Hoeschst nuclear stain (Cell Signaling Technology 4082S, 2 ug/mL). Samples were washed with washing buffer (three times, 5 min per wash) before being imaged using a confocal microscope (40x oil immersion, Leica SP8 confocal system). All images were processed using open-source Fiji software [35 (link), 36 (link)].
Mouse anti yap
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-YAP is a primary antibody that specifically binds to the YAP protein, a key transcriptional regulator involved in the Hippo signaling pathway. It can be used to detect and analyze the expression of YAP in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Rabbit anti phospho yap, by Cell Signaling Technology (2 mentions)
Rhodamine phalloidin, by Merck Group (1 mentions)
Sp8 confocal system, by Leica (1 mentions)
Mouse anti runx2, by Abcam (1 mentions)
Mouse anti β1 integrin, by Abcam (1 mentions)
Rabbit anti paxillin, by Abcam (1 mentions)
Rabbit anti p fak, by Abcam (1 mentions)
Formaldehyde, by Polysciences (1 mentions)
Mouse anti zo 1, by Thermo Fisher Scientific (1 mentions)
Draq5, by Cell Signaling Technology (1 mentions)
Mouse anti cdx2, by BioGenex (1 mentions)
Alexa flour 647, by Jackson ImmunoResearch (1 mentions)
Goat anti sox2, by Neuromics (1 mentions)
Rat anti cdh1, by Merck Group (1 mentions)
Fluoview fv1000 confocal laser scanning microscope system, by Olympus (1 mentions)
Mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Phalloidin atto 488, by Merck Group (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
13 protocols using mouse anti yap
1
Immunofluorescence Staining of Integrin Proteins
2
Embryo Immunostaining Protocol
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Embryos were fixed with 4% formaldehyde (Polysciences) for 10 min, permeabilized with 0.5% Triton X-100 (Sigma Aldrich) for 30 min, and then blocked with blocking solution (10% Fetal Bovine Serum (Hyclone), 0.1% Triton X-100) for 1 hr at room temperature, or overnight at 4°C. Primary Antibodies used were: mouse anti-CDX2 (Biogenex, CDX2-88), goat anti-SOX2 (Neuromics, GT15098), rabbit anti-PARD6B (Santa Cruz, sc-67393), rabbit anti-PARD6B (Novus Biologicals, NBP1-87337), mouse anti-PKCζ (Santa Cruz Biotechnology, sc-17781), rat anti-CDH1 (Sigma Aldrich, U3254), mouse anti-ZO1 (Thermo Fisher Scientific, 33–9100), mouse anti-YAP (Santa Cruz Biotechnology, sc101199), rabbit anti phospho-YAP (Cell Signaling Technologies, 4911), chicken anti-GFP (Aves, GFP-1020). Stains used were: Phallodin-633 (Invitrogen), DRAQ5 (Cell Signaling Technologies) and DAPI (Sigma Aldrich). Secondary antibodies conjugated to DyLight 488, Cy3 or Alexa Flour 647 fluorophores were obtained from Jackson ImmunoResearch. Embryos were imaged using an Olympus FluoView FV1000 Confocal Laser Scanning Microscope system with 20x UPlanFLN objective (0.5 NA) and 5x digital zoom. For each embryo, z-stacks were collected, with 5 µm intervals between optical sections. All embryos were imaged prior to knowledge of their genotypes.
3
Immunofluorescence Staining Protocol
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The following is a list of the primary antibodies used and their respective dilutions were: mouse anti-YAP, 1:200 (Santa Cruz, cat. no. sc-271134); mouse anti-CK20, 1:100 (Dako, cat. no. M7019); rabbit anti-VE-cadherin, 1:2000 (Invitrogen, cat. no. PA5-19612), rabbit anti-pMLC P-Myosin Light Chain 2 (Thr18/Ser19), 1:200 (Cell Signaling cat. no. 3674), rabbit anti-. Ezrin/Radixin/Moesin, 1:100 (Cell Signaling cat. no. 3142), rabbit anti-phospho Ezrin (Thr567)/ Radixin (Thr564)/ Moesin (Thr558), 1:100 (Cell Signaling cat. no. 3141). The secondary antibodies used were: goat anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific, cat. no. A-11029); donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, cat. no. A-21206), goat anti-rabbit Alexa Fluor 555 (Thermo Fisher Scientific, cat. no. A-21429) and goat anti-mouse Alexa Fluor 405 (Abcam, cat. no. ab175660). All the secondary antibodies were used at a dilution of 1:400. To label F-actin, phalloidin Atto 488 (Sigma-Aldrich cat. no. 49409) was used at 1:500 and phalloidin Alexa Fluor-647 (Thermo Fisher Scientific, cat. no. A22287) at 1:400. Hoechst (Thermo Fisher Scientific, cat. no. 33342) was used to label nuclei.
4
NEK1 Variant Generation and Analysis
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Wild type human full length NEK1 mammalian expression plasmid was purchased from Origene (MR216282). NEK1 T141A variant was generated by site-directed mutagenesis, as previously described [23 (link)]. Generation of His-tagged N-terminal NEK1 (aa 1–480) bacterial expression plasmid was conducted as previously described. Human full length MK5 bacterial expression plasmid was purchased from Vector Builder. The following antibodies were used in this study: mouse anti-YAP (Santa Cruz Biotechnology, SCBT, Dallas, TX, USA, cat# sc101199), rabbit anti-phospho-YAP (Cell Signaling Technology, CST, Dallas, TX, USA, cat# 13008), mouse anti-NEK1 (SCBT, cat# sc 398813, Dallas, TX, USA), rabbit anti-phospho-NEK1 pT141 (lab-generated), rabbit anti-phospho-tyrosine (CST, cat# 8954S, Dallas, TX, USA), HRP-conjugated anti-β-tubulin (SCBT, Dallas, TX, USA, cat# sc-23949), mouse IgG (SCBT, Dallas, TX, USA, cat# sc-2025), and rabbit anti-actin (Abcam, Cambridge, MA, USA, cat# ab1801).
5
Immunofluorescence Staining Protocol
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Samples were fixed after 4 or 24 h with 4% (w/v) paraformaldehyde (Merck, #1040051000). A standard staining procedure was performed with the following antibodies and dyes (final concentration): Rabbit anti-FN (1.2 µg/ml, Sigma, #F3648), rabbit anti-LN (5 µg/ml, Sigma, #L9393), mouse anti-YAP (0.33 µg/ml, Santa Cruz, #sc-101199; applied at 4 °C over night), goat anti-mouse Cy3 (7.5 µg/ml, Jackson ImmunoResearch, #115-165-166), goat anti-rabbit AF568 (7.5 µg/ml, Jackson ImmunoResearch, #111-165-144), goat anti-rabbit AlexaFluor (AF) 647 (7.5 µg/ml, Jackson ImmunoResearch, #111-606-045), DAPI (2–0.2 µg/ml, Roth, #6335.1), Phalloidin AF568 (1 U/ml, Invitrogen, #A12380), Phalloidin AF647 (2 U/ml, Invitrogen, #A22287).
6
Immunohistochemistry of Inner Ear Tissues
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Utricles were dissected in ice-cold HBSS and fixed in 4% formaldehyde for 1 hr at room temperature. Whole inner ears were fixed for 18 hr at 4˚C, treated with 0.88 M sucrose for 18 hr at 4˚C, embedded in Tissue-Tek O.C.T. (Sakura), and frozen in liquid-nitrogen vapor. Wholemounted sensory epithelia or 10 μm frozen sections were then blocked with 3% normal donkey serum (Sigma-Aldrich) in 500 mM NaCl, 0.3% Triton X-100 (Sigma-Aldrich), and 20 mM tris(hydroxymethyl)aminomethane (Bio-Rad) at pH 7.5. The primary antisera—goat anti-Sox2 (Santa Cruz), rabbit anti-Myo7A (Proteus Bioscience), rabbit anti-GFP (Torrey Pines Biolabs), mouse anti-Yap (Santa Cruz), and rabbit anti-Yap (Cell Signaling)—were reconstituted in blocking solution and applied overnight at 4˚C. For labeling with E-cadherin antibodies from clone DECMA-1 (Millipore) no Triton X-100 was used in the blocking solution.
Samples were washed with phosphate-buffered saline solution supplemented with 0.1% Tween 20 (Sigma-Aldrich), after which Alexa Fluor-labeled secondary antisera (Life Technologies) were applied in the same solution for 1 hr at room temperature.
Phalloidin conjugated to Alexa 633 was used to label filamentous actin and nuclei were stained with 3 μM DAPI.
Samples were washed with phosphate-buffered saline solution supplemented with 0.1% Tween 20 (Sigma-Aldrich), after which Alexa Fluor-labeled secondary antisera (Life Technologies) were applied in the same solution for 1 hr at room temperature.
Phalloidin conjugated to Alexa 633 was used to label filamentous actin and nuclei were stained with 3 μM DAPI.
7
Immunolabeling of Gustatory and Neural Markers
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The following antibodies were used: rabbit anti-METTL3 (1:200; Abcam, Cat No: ab195352), rabbit anti-KRT8 (1:200; Abcam, Cat No: ab53280), goat anti- gustducin (1:100; Aviva Systems Biology, Cat No: OAEB00418), rabbit anti-PGP9.5 (1:500, Thermo Fisher, Cat No: 480012), rabbit anti-SNAP25 (1:100; Proteintech, Cat No: 14903-1-AP), mouse anti- PAN-CK(1:100, Thermo Fisher, Cat No: MA1-82041), mouse anti-KRT13 (1:200; Abcam, Cat No: ab16112), rabbit anti-p63 (1:300; Abcam, Cat No: ab53039), mouse anti-LATS1 (1:100; Santa Cruz Biotechnology, Cat No: sc-398560), mouse anti-YAP (1:200; Santa Cruz Biotechnology, Cat No: sc-101199), mouse anti-TAZ (1:100; Santa Cruz, Cat No: sc-293183), rabbit anti-FZD7 (1:1 000 for western blot; Abcam, Cat No: ab64636), rabbit anti-β-catenin (1:250; Proteintech, Cat No: 51067-2-AP), rabbit anti-LEF1 (1:200; Abcam, Cat No: ab137872).
8
Immunofluorescence Analysis of Mouse Ovarian Cryosections
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To prepare cryosections, ovaries were fixed in freshly prepared 2% (w/v) paraformaldehyde (EMS Biosciences) in PBS at 4°C for 2 h, then washed through a sucrose gradient (10%, 20%, 30% in PBS), embedded in Tissue-Tek OCT (Sakura Finetek), and stored at −80°C. Sections were cut at 8 μm thickness and rehydrated for 10 min in PBS. Immunostaining was performed using Vector Mouse on Mouse Immunodetection Kit (2201), following the manufacturer's directions except that the PBS was supplemented with 0.05% Triton X-100 and 0.15% glycine. Slides were counterstained using 4′,6-diamidino-2-phenylindole and covered with coverslips using Vectashield (Vector) as an antifading solution. Slides were examined using a TCS-SP5 laser-scanning confocal microscope (Leica), and images were analyzed using LAS AF (Leica Microsystems CMS GmbH) and Imaris (Bitplane). Primary antibodies used were rabbit polyclonal anti-MVH (1:1000 dilution, ab13840; Abcam) and mouse anti-YAP (1:100 dilution, 101199; Santa Cruz Biotechnology). Paraffin-embedded sections were prepared and used for immunohistochemistry as previously described [42 ], using the same primary antibodies and dilutions as for immunofluorescence. YAP was detected using Alexa488-conjugated rabbit anti-mouse (1:500 dilution, A11059; Life Technologies). Images were recorded using an LSM 510 confocal microscope (Zeiss).
9
Temporal Analysis of YAP Phosphorylation
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Cells were lysed 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h after the sonication with 2X Laemmli Sample Buffer. The cell lysates were separated on 12.5% polyacrylamide gels and transferred for 2 h to a nitrocellulose membrane with 0.45 μm pore-size (neoLab Migge GmbH, Heidelberg, Germany). The membranes were blocked in TBST buffer (TBS buffer with 0.1% Tween-20 (Sigma-Aldrich, Munich, Germany)) at pH 8, containing 3% Bovine Serum Albumin (BSA) (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) for 1 h at room temperature. Then the membranes were incubated overnight at 4°C in either rabbit anti-phospho-YAP(Serine127) (Cell Signaling Technology, Inc., Danvers, MA, USA), or rabbit anti-GAPDH (Cell Signaling Technology, Inc., Danvers, MA, USA), or mouse anti-YAP (Santa Cruz Biotechnology, Dallas, TX, USA) antibody. The next day the membranes were washed 3 times in TBST buffer and incubated for 50 min at room temperature in TBST buffer containing 3% BSA and either IRDye® 800CW Goat (polyclonal) Anti-Rabbit IgG (H+L) or 680RD Goat (polyclonal) Anti-Mouse IgG (H+L), Highly Cross Adsorbed secondary antibody (LI-COR, Lincoln, NE, USA). The membranes were washed twice with TBST and once with TBS buffer. The near-infrared fluorescence signal was imaged and quantified by LI-COR Odyssey system.
10
Immunocytochemistry of Cell Signaling Proteins
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After 3 h in culture on the microprinted PDMS layers, cells were fixed with 4% PFA at room temperature (RT) for 15 min and then permeabilized with 0.2% Triton X-100 in PBS for 5 min at RT and washed 3 times in PBS. The immunochemistry process was performed following these steps: the fixed cells were incubated for 30 min in a blocking solution (0.2% tween, 1% Bovine Serum Albumine BSA, 1% Foetal Bovine Serum FBS) at RT, then the primary antibody was incubated for 40 min, washed three times in PBS-tween 0.2%, the secondary was incubated for 30 min, washed three times with PBS. The nucleus was then labeled with 4′,6-diamidino-2-phenylindole (DAPI) for 30 min at RT and the actin filaments (F-actin) with SiR-actin (Spirochrome) at 100 nM in PBS overnight at 4 °C. The antibodies used for the immunochemistry were: mouse anti-Flag (Sigma Aldrich M2 F3165) 1/1000, rabbit anti-SMYD3 (Abcam ab187149) 1/300, rabbit anti-TAZ (Cell Signaling Technology 4883) 1/500, mouse anti-YAP (Santa Cruz Biotechnology 101199) 1/250, rabbit anti-Pan tri-methyl lysine (Cell Signaling Technology #14680) 1/1000, rabbit anti-Pan di-methyl lysine (Cell Signaling Technology #14117) 1/1000, DNase 1 Alexa Fluor 594 (Life Technologies) 1/1000, Rabbit anti-SETDB1 (Santa Cruz Biotechnology sc-66884) 1/200.
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