Immunohistochemical staining was performed on paraffin embedded ccRCC tissues. After microtome sectioning (5 μm), the slides were stained with anti-GLUT1 (Proteintech), anti-HK2 (Proteintech), and anti-FBP1 (Sigma) antibodies. Immunohistochemical analyses were conducted by two experienced pathologists. The slides were scored according to staining intensity (0–3). Slides with a score of 2 or 3 were considered high expression, and slices with a score of 0 or 1 were considered low expression.
Anti glut1
Manufactured by Proteintech
Sourced in United States
Anti-GLUT1 is a laboratory reagent used to detect and quantify the GLUT1 glucose transporter protein. It is a specific antibody that binds to the GLUT1 protein, allowing for its identification and analysis in various biological samples.
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Lab products found in correlation
Anti ldha, by Proteintech (4 mentions)
Anti hk2, by Proteintech (3 mentions)
Anti hif 1α, by Proteintech (3 mentions)
Anti β actin, by Proteintech (3 mentions)
Anti gapdh, by Proteintech (2 mentions)
Anti pgk1, by Proteintech (2 mentions)
Anti pkm2, by Proteintech (2 mentions)
Anti glut3, by Proteintech (2 mentions)
Anti fbp1, by Merck Group (1 mentions)
Anti il 17a, by Proteintech (1 mentions)
Anti adipor1, by Abcam (1 mentions)
Anti β actin, by Abbkine (1 mentions)
Anti p lats1 ser909, by Cell Signaling Technology (1 mentions)
Anti yap, by Proteintech (1 mentions)
Anti yap, by Cell Signaling Technology (1 mentions)
Anti p stat3, by Cell Signaling Technology (1 mentions)
Anti β actin, by MultiSciences Biotech (1 mentions)
Anti gfap, by Cell Signaling Technology (1 mentions)
Anti pdk1, by Proteintech (1 mentions)
Pv 9000, by ZSGB-BIO (1 mentions)
13 protocols using anti glut1
1
Immunohistochemical Analysis of Metabolic Enzymes in ccRCC
2
Quantification of Protein Expression
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An equal amount of protein (30 μg) was analyzed per sample using SDS polyacrylamide gel electrophoresis. Separated proteins were then transferred to a PVDF membrane. The membrane was incubated with primary antibodies overnight at 4°C with gentle shaking. The membranes were washed and incubated with a secondary antibody, followed by electrochemical luminescence detection. Relative protein levels were quantified by scanning densitometry and analyzed using ImageJ software. Protein detection was carried out using anti-GAPDH, anti-β-actin, anti-HIF-1α, anti-IL-17A, anti-GLUT1, anti-HK2 (proteintech) and anti-AdipoR1 (Abcam) Abs.
3
Protein Expression Analysis in Tumors
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We performed immunohistochemical staining and western blotting using methods that have been described previously [24 (link)]. Anti-YAP (1:100, immunohistochemistry), Anti–phosphorylated-YAP (p-YAP) (Ser127) (1:1000, western blot), and anti–p-LATS1 (Ser909) (1:1000, western blot) were from Cell Signaling Technology (Shanghai, China). Anti-YAP (1:1000, western blot), anti-LDHA (1:1000, western blot), anti-PKM2 (1:1000, western blot), anti-Glut1 (1:1000, western blotting), anti-PGK1 (1:1000, western blot), anti–large tumor suppressor kinase 1 (LATS1, 1:1000, western blotting), and anti–HIF-1α (1:1000, western blot) rabbit monoclonal antibodies were from Proteintech (Wuhan, China). Anti–matrix metalloproteinase 2 (MMP2, 1:1000, western blot), anti-MMP9 (1:1000, western blot), horseradish peroxidase–linked secondary antibody (1:5000, western blot), and anti–β-actin (1:3000, western blot) were from Abbkine (Wuhan, China). The protein bands were analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Spinal Cord Proteins
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The procedure was like the method described previously (30 (link)). The rats were killed under anesthesia. About 100 mg of lumbar spinal cord tissue was cut into pieces. After adding protein lysate into a homogenizer, the homogenate was ground, and centrifuged supernatant was the total protein extracted. Then we used the BCA method to determine the protein concentration of each group. Each sample was loaded with 30 µg and boiled with a loading buffer for 15 minutes. Then 10% of SDS-PAGE gel electrophoresis was used to isolate the protein and transferred to the PVDF membrane. The 5% skim milk powder was sealed at RT for two hours. Then the first Ab (anti GFAP 1:1,000, #3670, Cell Signaling Technology, anti-p-STAT3 1:1,000, #9145, Cell Signaling Technology, anti-PKM2 1:1,000, 15822-1-AP, Proteintech, anti-GLUT1, 1:1,000, 21829-1-AP, Proteintech, anti-Lamin B1 1:1,000, MULTISCIENCES, anti-β-actin 1:1,000, MULTISCIENCES) were incubated overnight at 4 °C. On the second day, TBST was used to wash the membrane 3×10 minutes, and the corresponding horseradish peroxidase-labeled secondary Ab (1:4,000) was added respectively, incubated on the shaker for 2 hours. Finally, ECL chromogenic fluid was added for exposure, and the image was taken and analyzed in the gel imaging system, and each band was calculated with Image J software.
5
Immunohistochemical Analysis of Tumor Markers
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Tumour tissues were fixed with 4% paraformaldehyde and cut into 4‐μm‐thick sections. Immunohistochemistry (IHC) was performed according to the protocol. The sections were incubated with rabbit anti‐FOXM1 (1:100, 20,459, CST), anti‐PDK1 (1:100, 18,262‐1‐AP; Proteintech), anti‐GLUT1 (1:200, 66,290‐1‐Ig; Proteintech), anti‐ LDHA (1:200, 66,287‐1‐Ig; Proteintech) and anti‐HK2 (1:100, 22,029‐1‐AP; Proteintech) antibodies overnight at 4°C. The sections were then sequentially incubated with a biotinylated secondary antibody (PV‐9000; ZSGB‐Bio) to detect the primary antibodies. Images were obtained using TissueFAXS systems (TissueGnostics).
6
Western Blot Analysis of Protein Targets
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Total protein extracts were obtained using the RIPA lysis buffer (Sigma, USA). Protein concentration was measured using a BCA Protein Assay kit (Pierce, USA). Protein samples were resolved in 7.5 and 15% SDS-PAGE gels and transferred to Immuno-blot PVDF Membrane (Bio-Rad, USA). The membranes were blocked using 5% non-fat milk in Tris-buffered saline with Tween-20. The membranes were then incubated with primary antibodies followed by incubation with secondary peroxidase labeled anti-rabbit or anti-mouse antibodies (Santa Cruz Biotechnology, USA). The protein signals were detected using an enhanced chemiluminescent solution (Millipore, USA). Primary antibodies used include anti-tubulin, anti-actin (Sigma, USA), anti-PFKP, anti-p53, anti-phospho-p53, anti-p21, anti-ACC, anti-phospho-ACC, anti-AMPK, anti-phospho-AMPK (Cell Signaling Technology, USA), anti-PFKM, anti-PFKL, anti-GLUT1 and anti-V5 (Proteintech Group, USA).
7
Western Blot Analysis of Protein Expression
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Total proteins were extracted using RIPA buffer (Boster, Wuhan, China) with a protease inhibitor PMSF (Boster, Wuhan, China). BCA method was used to determine the protein levels in cells. Subsequently, the protein samples were separated using 10% SDS-PAGE and transferred onto a PVDF membrane. This was followed by blocking with 5% fat-free milk (Servicebio, Wuhan, China) at room temperature for 2 hours, and incubation of membranes overnight at 4°C with the following primary antibodies diluted at 1:1000: anti-HIF1α, anti-MCL1, anti-VEGF, anti-Glut1 and β-actin (Proteintech, Wuhan, China). The membranes were rinsed in TBST three times, and then incubated with horseradish peroxidase (HRP) conjugated rabbit or mouse secondary antibodies for 2 hours. Densitometric quantification was performed using the Image J software (version 1.50i) to analyse intensity of the protein bands.
8
HHV-6A Infection: Protein Expression
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HHV-6A-infected and mock-infected cells were collected at 24, 48 and 72 h post-infection. Cells were lysed in cell lysis buffer at 4°C for 30 min. Protein concentrations of the cell lysates were measured using the BCA reagent (Beyotime Biotechnology). Equivalent amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (BIO-RAD) and detected with the corresponding primary and secondary antibodies. The protein bands were visualized using the enhanced chemiluminescence (ECL) system (Tanon Science & Technology). The antibodies used in Western-blotting analyses included anti–mTOR, anti–phospho-mTOR, anti-p70S6K, anti–phospho-p70S6K, anti-4E-BP1, anti–phospho-4E-BP1, anti-AKT, anti-phospho-AKT, anti-TSC2, anti-phospho-TSC2 (Cell Signaling Technology), anti-β-actin, anti-Glut1 and anti-Glut3 (Proteintech Biotechnology). A monoclonal antibody against the HHV-6 IE1 and gB was produced by our laboratory.
9
Immunohistochemical Evaluation of Glycolytic Enzymes
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The IHC staining was used to examine the expression levels of c-Myc and glycolytic enzymes. The paraffin-embedded sections were incubated in a pressure cooker containing antigen retrieval buffer (pH 6.0) (Solarbio; G1202). Next, the slides were uniformly covered with 3% bovine serum albumin (Solarbio; A8020), and probed with the following primary antibodies prepared in a wet box at 4 °C overnight: anti-c-Myc (Servicebio; GB13076), anti-GLUT1 (Proteintech, 21,829–1-AP), anti-ENO1 (Proteintech, 11,204–1-AP), anti-PGK1 (Proteintech, 17,811–1-AP), anti-ALDOA (Proteintech, 11,217–1-AP), anti-HK1 (Proteintech, 19,662–1-AP), and anti-LDHA (Proteintech, 19,987–1-AP) antibodies. Next, the horseradish peroxidase (HRP)-labeled secondary antibody was used for 50 min. Then, the sections were then counterstained with hematoxylin.
The stained sections were scanned using Pannoramic MIDI and analyzed using Quant Center, which automatically identified all the strongly positive, moderately positive, weakly positive, and negative areas in the tissue sections. The H-scores were calculated as ∑ (percentage of intensity × intensity). The staining intensity was divided into three categories—strong, moderate, and weak—and the corresponding score was 3, 2, and 1, respectively.
The stained sections were scanned using Pannoramic MIDI and analyzed using Quant Center, which automatically identified all the strongly positive, moderately positive, weakly positive, and negative areas in the tissue sections. The H-scores were calculated as ∑ (percentage of intensity × intensity). The staining intensity was divided into three categories—strong, moderate, and weak—and the corresponding score was 3, 2, and 1, respectively.
10
Western Blotting for Autophagy and Metabolism Markers
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