The paraffin sections of tumor tissues from colorectal cancer were obtained from Tianjin Medical University Cancer Institute and Hospital. The sections were then deparaffinized and rehydrated in alcohol and water. Antigen retrieval was undertaken in sodium citrate buffer for 5 minutes at 100℃. The sections were then incubated with anti‐CD68 (1:300; Abcam), anti‐TGF‐β1 (1:100, Ab215715; Abcam), anti‐Wnt3A (1:200, Ab219412; Abcam), anti‐TRIB3 (1:500, Ab137526; Abcam), and anti‐HIF1α (1:300, Ab179483; Abcam) primary Abs at 4℃ overnight. For immunofluorescent staining, the samples were incubated with goat anti‐rabbit secondary Abs (1:1000; Thermo Fisher Scientific) and the nuclei were stained with DAPI (Solarbio). Images were obtained using a laser scanning confocal microscope (Leica). For immunohistochemical staining, samples were incubated with an ABC HRP Kit (Thermo Fisher Scientific) and counterstained with hematoxylin (Solarbio). The intensity of protein expression was calculated by ImageJ 2.0 (immunofluorescence) and Image‐Pro Plus 7.0 (immunohistochemistry) software. The mean of protein expression intensity in 30 fields was determined in each sample. Fifteen samples from 15 colorectal cancer patients were included in each group.
Ab137526
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab137526 is an antibody that can be used as a tool for research applications. It is a polyclonal antibody that specifically recognizes the target protein. The antibody can be used in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab219412, by Abcam (2 mentions)
Ab179483, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Hematoxylin, by Solarbio (1 mentions)
Ab215715, by Abcam (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Abc hrp kit, by Thermo Fisher Scientific (1 mentions)
Anti cd68, by Abcam (1 mentions)
Alexa fluro 594, by Thermo Fisher Scientific (1 mentions)
Alexa fluro 488, by Thermo Fisher Scientific (1 mentions)
Ab171380, by Abcam (1 mentions)
Ab52857, by Abcam (1 mentions)
Ab955, by Abcam (1 mentions)
Laser scanning confocal imaging system, by Olympus (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab91526, by Abcam (1 mentions)
Ab51520, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
6 protocols using ab137526
1
Immunohistochemical Analysis of Colorectal Cancer
2
Immunofluorescence Analysis of Breast Cancer
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The frozen primary or PDX breast cancer tissues were fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, blocked with 3% BSA, and stained with an anti-TRIB3 (1:100, Abcam, ab137526), anti-CD68 (1:100, Abcam, ab955), anti-FOXO1 (1:100, Abcam, ab52857), or anti-SOX2 (1:100, Abcam, ab171380) primary antibody followed by Alexa Fluro 488 and/or Alexa Fluro594 secondary antibodies (1:200, Life Technologies). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole. Fluorescence images were obtained by using a laser scanning confocal imaging system (Olympus Microsystems).
3
Dissecting Autophagy Signaling in Glioma
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Glioma and normal tissues were minced, homogenized, and digested in RIPA lysis buffer (with protease and phosphatase inhibitors, Thermo Scientific). Cells were scraped off culture plates on ice and lysed with RIPA (with protease and phosphatase inhibitors, Thermo Scientific). The resulting suspensions were centrifuged at 13,000 rpm for 20 minutes at 4°C, and the protein supernatant was collected. Protein samples were prepared for PAGE gel electrophoresis. Proteins were then transferred to a PVDF membrane (BioRad) for immunoblotting with relevant antibodies. The following antibodies were used in this study: TRIB3 (ab137526, Abcam), P62 (ab91526, Abcam), LC3 (ab51520, Abcam) and ATG5 (#15071, Cell Signaling), ATG7 (ab53255, Abcam) and GAPDH (#5174, Cell Signaling) served as loading controls.
4
Immunohistochemistry protocol for TRIB3 and FABP1
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IHC was performed mainly as previously described (Yang et al., 2018 (link)). 4-µm-thick tissues were mounted in poly-l-lysine-coated glass slides and baked overnight at 65°C. After deparaffinization with xylene and hydration with graded ethanol, tissue sections were heated in EDTA buffer for 20 min for antigen retrieval. The 10% normal goat serum was used for incubation for 15 min to reduce non-specific binding. Then sections were then incubated with primary antibody for 60 min at room temperature (24–27°C) and secondary antibody for 10 min. At last, the slides were stained with DAB (DAB-1031, Maxim Inc., Fujian, China) and counterstained with hematoxylin. The anti-TRIB3 (ab-137526, 1:1,000 dilution; Abcam, Cambridge, United Kingdom) and anti-FABP1 (ab-171739, 1:4,000 dilution; Abcam, Cambridge, United Kingdom) antibodies were used in our experiment.
5
Western Blot Analysis of Cellular Proteins
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The protein lysates from CT26 and HCT116 cells were separated by SDS‐PAGE and then transferred to a PVDF membrane (Millipore). The membrane was incubated with the primary Abs against anti‐HIF1α (1:1000, Ab179483; Abcam), anti‐TRIB3 (1:1000, Ab137526; Abcam), anti‐β‐catenin (1:1000, Ab32572; Abcam), anti‐Wnt3A (1:1000, Ab219412; Abcam), and anti‐β‐actin (1:1000, Ab8226; Abcam), followed by incubation with an HRP‐conjugated secondary Ab (1:1000; Abcam).
6
TRIB3 Protein Expression Analysis by Western Blot
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Western blot was used to assess TRIB3 protein expression as previously described (18 (link)). Briefly, whole cell lysates were prepared and quantified using RIPA reagent. Protein samples were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were incubated in a blocking solution, followed by incubation with rabbit anti human monoclonal antibody against TRIB3 (1:1,000, ab137526, Abcam, USA), SMARCD3 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily D, member 3; 1:1,000, ab171075,Abcam, USA), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000, Abcam, USA) for 18 hours at 4 °C. Membranes were then washed three times with Tris-buffered saline Tween (TBST) and incubated with a goat anti-rabbit IgG-AP secondary antibody (1:5,000 dilution, #HA1019, HuaBio, Hangzhou, China) in room temperature for 1 hour. After washing, the proteins on the membrane were detected by an enzyme labeling method and chemiluminescence in accordance with the manufacturer’s instructions (Omni ECL reagent (EpiZyme, Shanghai, China).
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