Mouse tissues were fixed with 4% formalin and embedded in paraffin. Immunohistochemical studies were conducted using 5-µm sections of paraffin-embedded tissue mounted on precoated slides. The tissue was dewaxed with xylene and rehydrated through graded alcohol according to standard procedures. After antigen retrieval in citrate buffer for 10 min in the microwave at power level 7, the endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 1 h. Blocking was done using Rodent Block M (Nobel Biocare). Slides were incubated with various antibodies with the working conditions detailed below. Secondary conjugation was done with HRP polymer (Nobel Biocare) specific for rabbits or mice. Staining was developed with a DAB kit (Vector Laboratories), and counterstaining was done with hematoxylin. The antibodies used were PRKCI (1:250; Sigma HPA026574), human YAP1 (1:250; Cell Signaling, 4912), mouse YAP1 (1:250; Novus Biologics, NB110-58358), Ki-67 (Dako), TP53 (1:500; Calbiochem, Ab-6), PAX8 (1:500; Proteintech, 10336), PAX2 (1:500; Invitrogen, 716000), and TNFα (1:200; Abcam, T8300).
Human yap1
Manufactured by Cell Signaling Technology
Human YAP1 is a recombinant protein that represents the full-length human Yes-associated protein 1 (YAP1). YAP1 is a transcriptional coactivator that regulates cell growth and apoptosis in response to various cellular signals.
Automatically generated - may contain errors
Lab products found in correlation
Dab kit, by Vector Laboratories (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Alpha imager, by Bio-Techne (1 mentions)
Proteojet mammalian cell lysis reagent, by Thermo Fisher Scientific (1 mentions)
1 step nbt bcip reagent, by Thermo Fisher Scientific (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Goat anti rabbit or anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
Phosstop, by Roche (1 mentions)
C myc, by Abcam (1 mentions)
Survivin, by Abcam (1 mentions)
2 protocols using human yap1
1
Paraffin-Embedded Tissue Immunohistochemistry
2
Western Blot Analysis of Key Cell Signaling Proteins
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Lysates were obtained from 1 × 106 cultured cells with a mixture of ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Inc.), phenylmethanesulfonyl fluoride (Roche, Inc.) and PhosSTOP (Roche, Inc.). About 20 μg protein was extracted from each sample and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After the membrane was blocked in 5% nonfat milk, the protein of interest was probed with the appropriate antibody: human YAP1 (1:1000; Cell Signaling Technology), c-MYC (1:1000; abcam), Survivin (1:1000; abcam), KI67 (1:1000; Cell Signaling Technology), PCNA (1:1000; Cell Signaling Technology), ERK1/2 (1:1000; Cell Signaling Technology), p-ERK1/2 (1:1000; Cell Signaling Technology), AKT (1:1000; Cell Signaling Technology), p-AKT (1:1000; Cell Signaling Technology), or GAPDH (1:5000; Abcam). Then, the membrane was incubated with goat anti-rabbit or anti-mouse IgG (1:5000 for both; Jackson ImmunoResearch Laboratories) and treated with enhanced chemiluminescence reagents (Thermo Fisher Scientific). The bands were visualized with 1-stepTM NBT/BCIP reagents (Thermo Fisher Scientific, Rockford, IL, USA) and detected with Alpha Imager (Alpha Innotech, San Leandro, CA, USA).
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