Fixed tissue samples were sectioned at 5 μm thickness, deparaffinized and rehydrated. Then slides were placed in a solution containing 0.3% hydrogen peroxide at room temperature for 10 min and subjected to heat-induced antigen retrieval in EDTA buffer (PH 8.0) for 8 min in a microwave oven. Nonspecific staining was prevented with 10% goat serum incubation for 30 min. Afterwards, the slides were incubated at 4°C overnight with YAP (D8H1X) XP® Rabbit mAb (dilution 1:100) (#14074, Cell signaling, Danvers, MA, USA) and then with secondary antibody avidin-biotin-peroxudase-conjugated IgG at room temperature for 30 min. Proteins were visualized according to the manufacturer's instruction using 3,3’-diaminobenzidine as the substrate. The IHC results were assessed by two independent pathological investigators blinded to the patients’ and clinical status. Two independent qualified experts evaluated YAP1 expression simultaneously.
Yap d8h1x xp rabbit mab
Manufactured by Cell Signaling Technology
Sourced in United States
The YAP (D8H1X) XP® Rabbit mAb is a primary antibody that specifically recognizes the Yes-associated protein (YAP) in various species. YAP is a transcriptional regulator involved in the Hippo signaling pathway, which plays a critical role in cell growth, proliferation, and organ size control.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Mouse anti vinculin hvin 1, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Mayer s hematoxylin staining solution, by Merck Group (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Peroxidase conjugated rabbit anti mouse igg or goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Immobilon classico or forte substrate, by Merck Group (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Amersham imager 680, by Cytiva (1 mentions)
Gapdh monoclonal antibody, by Proteintech (1 mentions)
Alpha tubulin, by Proteintech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
6 protocols using yap d8h1x xp rabbit mab
1
Immunohistochemical Analysis of YAP1 Expression
2
Immunostaining of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell were fixed in 4% (vol/vol) paraformaldehyde (Alfa-Aeser) for 15 min at RT, permeabilized with 0.1% (vol/vol) Triton X-100 (EMD Millipore) for 3 min, and blocked with 1% (vol/vol) bovine serum albumin (BSA; Thermo Fisher) for 30 min in a humid chamber. Cells were incubated with primary antibodies for 2 h, rinsed with 1% BSA in PBS, and then incubated with secondary antibodies and phalloidin (3:500; Thermo Fisher Scientific) for 30 min in a humid chamber. The following primary antibodies were used for immunostaining: mouse anti-vinculin hVin-1 (1:1000; Sigma) and YAP (D8H1X) XP Rabbit mAb (1:1000; Cell Signaling). As secondary antibodies we used Alexa Fluor 488 goat anti-mouse (1:500) and Alexa Fluor 488 goat anti-rabbit (1:500), both from Life Technologies. Finally, cells were incubated with NucBlue Live ReadyProbes Reagent (Thermo Fisher Scientific) for 20 min in PBS to stain nuclei. Samples were rinsed in PBS and stored in PBS at 4°C in the dark.
3
Immunohistochemistry Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EnVision™ FLEX+, mouse, high pH, (Link) immunohistochemistry kit (Dako North America company, K8002); antibody diluent (Dako North America company, s3022); Mayer's hematoxylin staining solution (Sigma Aldrich company, SLBT4555); Met (DLC2) XP rabbit mAb (Cell Signaling company, #8198); YAP (D8H1X) XP rabbit mAb (#14074; Cell Signaling Technology).
4
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used: YAP (D8H1X) XP Rabbit mAb (Cell Signaling Technologies, No. 14074, Danvers, MA, USA), phosphor-YAP (Ser127, D9W2I) Rabbit mAb (Cell Signaling Technologies, No. 13008, Danvers, MA, USA), CD146/MCAM Rabbit polyclonal antibody (Fisher, 17564-1-AP, Hampton, NH, USA). Additionally, AlexaFluor Phalloidin 488 (Invitrogen, No. A12379, Waltham, MA, USA) was used to visualize the actin cytoskeleton, ProLong Gold Antifade Mountant with DAPI (Invitrogen, No. P36935, Waltham, MA, USA) to visualize nuclei.
5
Western Blot Analysis of Protein Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells were lysed with RIPA buffer (25 mmol/L Tris‐Cl pH 7.6, 150 mmol/L NaCl, 1% NP‐40, 1% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (04080‐24; Nacalai Tesque). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (23225; Thermo Fisher Scientific). Whole cell extracts were mixed with 2× sample buffer (125 mmol/L Tris‐Cl pH 6.8, 4% SDS, 20% glycerol, 3.1% DTT, and 0.02% bromophenol blue) and heated for 5 minutes at 95°C. Whole cell extracts containing equal amounts of protein were separated by SDS‐PAGE and transferred to PVDF membranes (10600023; Cytiva). The following Abs were used: monoclonal anti‐ß‐actin clone C‐15 (A5441; Merck), YAP (D8H1X) XP rabbit mAb (#14074; Cell Signaling Technology), GATA‐3 (D13C9) XP Rabbit mAb (#5852; Cell Signaling Technology), Phox2a (37K‐2) (sc‐81978; Santa Cruz Biotechnology), Phox2b (B‐11) (sc‐376997; Santa Cruz Biotechnology), and peroxidase‐conjugated rabbit anti‐mouse IgG or goat anti‐rabbit IgG (315‐035‐048 or 111‐035‐144; Jackson ImmunoResearch). Chemiluminescence was developed using the Immobilon Classico or Forte substrate (WBLUC0100 or WBLUF0100; Merck) and detected on the Amersham Imager 680 (Cytiva).
6
Protein Expression Analysis by Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting assays were used to identify the different expressions of protein. Modified RIPA lysis buffer was used to collect the total cell lysates. BCA Protein Assay Kit (Beyotime, Jiangsu, China) was used to standardize the concentration of different samples. Proteins were separated by SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane by using Mini-PROTEAN® Tetra (Bio-Rad, Hercules, CA, USA), and probed with primary antibodies respectively. The primary antibodies were as follows: YAP (D8H1X) XP® Rabbit mAb (1:1000, 14074S, Cell signaling technology), SOX2 Polyclonal antibody (1:1000, 11064-1-AP; Proteintech), OCT4 Polyclonal antibody (1:1000, 11263-1-AP; Proteintech), NANOG Polyclonal antibody (1:1000, 14295-1-AP; Proteintech), CD44 Polyclonal antibody (1:2000, 15675-1-AP; Proteintech), CD133 Polyclonal antibody (1:2000, 18470-1-AP; Proteintech), GAPDH Monoclonal antibody (1:50000, 60004-1-lg; Proteintech), Alpha Tubulin Polyclonal antibody (1:2000, 11224-1-AP; Proteintech). Subsequently the membranes were incubated with corresponding secondary antibodies: HRP-Goat anti-rabbit IgG (H+L) (RS0002; Immunoway); HRP-Goat anti-mouse IgG (H+L) (RS0001; Immunoway). Blots were detected using an enhanced chemiluminescence system (S6, manufactured by CLiNX, Shanghai, China).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!