The tissues were deparaffinized in xylene and rehydrated in graded alcohols. First, 3% H2O2 was used to block endogenous peroxidase, followed by microwave retrieval. Tissue sections were blocked in 10% goat serum (Zhongshan Chemical Co., Beijing, China) and incubated consecutively with primary antibodies and a secondary antibody. The following primary antibodies were used: antibodies against eIF4E (1:50, sc-9976) from Santa Cruz, RIPK1 (1:50, Proteintech), VE-cadherin (1:100, ab33168), vimentin (1:200, ab92547), phospho-AKT (S473) (1:50, T40067F) from Abmart, E-cadherin (1:200, #3195) from Cell Signaling Technology, MMP-2 (1:250, 10373-2-AP) from Proteintech and MLKL(1:100, sc-293201) from Santa Cruz. DAB staining was performed for appropriate durations, and all sections were counterstained with hematoxylin. PBS was used in place of the primary antibodies for all negative controls. The staining intensity was scored as follows: 0 (negative), 1 (weak), 2 (medium) and 3 (high). The percentage of stained cells in the whole field was scored as follows: 0 (negative), 1 (≤25%), 2 (25–50%), and 3 (>50%). The final score was determined by adding the scores of the staining intensity and percentage of stained cells together.
Sc 9976
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-9976 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is a device designed for general laboratory use. The core function of Sc-9976 is to perform specific tasks required in a research or testing environment. No further details on the intended use or specifications of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Cell Signaling Technology (3 mentions)
Ve cadherin, by Proteintech (1 mentions)
Sc 293201, by Santa Cruz Biotechnology (1 mentions)
Ripk1, by Proteintech (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nel105001ea, by PerkinElmer (1 mentions)
Mabe343, by Merck Group (1 mentions)
Sc 713, by Santa Cruz Biotechnology (1 mentions)
Eif4e, by Santa Cruz Biotechnology (1 mentions)
Ripk1, by Santa Cruz Biotechnology (1 mentions)
C digit blot scanner, by Gene Tech (1 mentions)
Sc 47724, by Santa Cruz Biotechnology (1 mentions)
Ve cadherin, by Abcam (1 mentions)
Vimentin, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab31217, by Abcam (1 mentions)
Ab31218, by Abcam (1 mentions)
Myogenin, by Developmental Studies Hybridoma Bank (1 mentions)
Pdcd4, by Cell Signaling Technology (1 mentions)
5 protocols using sc 9976
1
Immunohistochemical Profiling of Cellular Markers
2
Western Blot Analysis of Muscle Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot assays were carried out with gastrocnemius muscles or C2C12 cells homogenized in RIPA buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, and 0.1% SDS]. Proteins were separated on a 4–12% Bis‐Tris gel (Life Technologies) and transferred onto PVDF membranes. The membranes were incubated with the following primary antibodies at 4°C overnight: anti‐IGF1 receptor (IGF1R) (1:1000; sc713, Santa Cruz), anti‐eIF4E (1:2000; sc9976, Santa Cruz), anti‐phospho‐eIF4E(Ser209) (1:2000; 9741, Cell Signaling), anti‐4EBP1 (1:2000; 9452, Cell Signaling), anti‐phospho‐4EBP1(Ser65) (1:1000; 9451, Cell Signaling), anti‐AKT (1:2000; 9272, Cell Signaling), anti‐phospho‐AKT (Ser473) (1:2000; 4060, Cell Signaling), anti‐puromycin (1:25000; MABE343, Sigma), anti‐eIF3M (1:1000; NBP1‐31045, Novus Biologicals), anti‐eIF4G (1:2000; 2498, Cell Signaling), anti‐eIF4B (1:1000; 3592, Cell Signaling), anti‐eIF2B5 (1:1000; 3595, Cell Signaling), and anti‐phospho‐eIF2α (Ser51) (1:1000; 9721, Cell Signaling). The membranes were next incubated with horseradish peroxidase‐conjugated secondary antibodies to the appropriate IgG (1:2000; Life Technologies) for 90 min. Bound antibodies were visualized with enhanced chemiluminescence reagents (NEL105001EA, PerkinElmer).
3
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted using SDS lysis buffer and transferred to PVDF membranes. After the membranes were blocked with 5% skim milk for 1 h, they were incubated with primary antibodies overnight at 4 °C, which was followed by incubation with secondary antibodies for 2 hours. Bands were visualized using a C-Digit Blot Scanner (Gene Company) and analyzed with ImageJ software. GADPH (1:1000, sc-47724, Santa Cruz) was used as a protein loading control. The following primary antibodies were used: antibodies against eIF4E (1:200, sc-9976) from Santa Cruz, RIPK1 (1:500, ab106393), VE-cadherin (1:500, ab33168), vimentin (1:1000, ab92547), and Snail (1:1000, ab180714) from Abcam (Cambridge, USA), phospho-AKT (S473) (1:500, T40067F) from Abmart, AKT (1:500, #AF6259) from Affinity, E-cadherin (1:1000, #3195) from Cell Signaling Technology, and MMP-2 (1:500, 10373-2-AP) from Proteintech.
4
Immunofluorescence Analysis of Epithelial-Mesenchymal Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were plated on coverslips, incubated at 37 °C overnight, permeabilized with 0.1% Triton X-100 and blocked with 5% FBS. Then, the cells were incubated with primary antibodies against eIF4E (sc-9976; Santa Cruz; 1:100), E-cadherin (#3195; Cell Signaling Technology; 1:100), and vimentin (ab92547; Abcam; 1:100). After incubation with fluorophore-conjugated secondary antibodies, the nuclei were counterstained with DAPI (Sigma). Images were acquired by fluorescence microscopy.
5
Western Blot Analysis of Translation Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were fractionated on SDS-polyacrylamide gels, and transferred to PVDF membranes (Bio-Rad). Antibodies used in this study were directed against: eIF4AI (ab31217; Abcam), eIF4AII (ab31218; Abcam), eIF4E (sc9976; Santa Cruz Biotech), eIF4GI (A300-502A; Bethyl Labs), PDCD4 (9535; Cell Signaling Tech), myogenin (F5D; Developmental Studies Hybridoma Bank), MyoD (M-318, sc-760, Santa Cruz Biotech), GAPDH (ab8245; Abcam), and β-actin (A5441; Sigma). 35S-methionine/cysteine protein labeling was performed as described previously [7] (link).
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!