Protein samples were handled with SDS-PAGE gels electrophoresis and transferred to PVDF membranes. PVDF membranes were incubated with primary S100A8 and GAPDH antibody (ab92331 and ab181602, Abcam, Cambridge, MA) and secondary antibody by turns, then treated with a chemiluminescence detection kit (Gene, Hong Kong, China). ImageJ software (BD, Franklin Lakes, NJ) was used to quantify the protein expression.
Ab92331
Manufactured by Abcam
Sourced in United Kingdom
Ab92331 is a laboratory equipment product. It is a device used for a specific function in a laboratory setting. No further details about the product's core function or intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab92507, by Abcam (4 mentions)
Ab105472, by Abcam (2 mentions)
Ab137867, by Cell Signaling Technology (2 mentions)
Ab92536, by Abcam (2 mentions)
Ac026, by ABclonal (2 mentions)
Ripa lysis buffer, by Applygen (2 mentions)
Chemiluminescence detection kit, by Gene Tech (1 mentions)
Ab181602, by Abcam (1 mentions)
Imagej software, by BD (1 mentions)
Ab133357, by Abcam (1 mentions)
Ab156008, by Abcam (1 mentions)
Ab225732, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ab183685, by Abcam (1 mentions)
Ab63818, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab125011, by Abcam (1 mentions)
Ab37657, by Abcam (1 mentions)
Ab2788, by Abcam (1 mentions)
Ab92726, by Abcam (1 mentions)
9 protocols using ab92331
1
SDS-PAGE and Immunoblot Analysis
2
Immunohistochemical Analysis of Mouse Tumor Samples
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Tissue specimens or tumor issues from mouse models were fixed in 4% PFA and embedded in paraffin. Immunohistochemistry and immunofluorescence were performed following a standard procedure5 . 5 μm sections were stained with hematoxylin and eosin (H&E) and incubated with primary antibodies including: anti-Ki67 (#ab16667, Abcam, 1:200), anti-CXCL1 (#GTX31184, GeneTex, 1:2000), anti-CXCL2 (#GTX31171, GeneTex, 1:1000), anti-CXCL8 (#T5153, ImmunoWay, 1:500), anti-CD11b (#ab133357, Abcam, 1:1000), anti-Gr-1 (#108401, Biolegend, 1:50), anti-MRP8 (#ab92331, Abcam, 1:2000), anti-CD4 (#ab183685, Abcam, 1:500), anti-CD8 (#98941, CST, 1:200), anti-TBX3 (#16741-1-AP, Proteintech, 1:200), anti-Thyroglobulin (#ab156008, Abcam, 1:400), anti-CXCR2 (#ab225732, Abcam, 1:100), anti-Gr-1 (#550291, BD, 1:100). IHC slides were scanned with a light microscope. The immunoreactive score (IRS Score) was calculated by multiplying percentage of positive cells and immunostaining intensity24 (link). The final score yielded a range from 0 to 12: 0-1 was considered to be no expression, 2–4 was low expression, 5–8 was moderate expression, while >9 was high expression. Tissue specimens of normal tissues, pathological grade I, II, III, and IV were grouped into Normal, Grade low (I and II) and Grade high (III and IV) for a few of grade II according to the pathological criteria of PTC.
3
Protein Expression Analysis by Western Blot
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Cells were lyzed in radioimmunoprecipitation assay buffer and lysates were assayed by western blot analysis for the expression of S100A8 (Abcam, Cambridge, UK, ab92331), S100A9 (Abcam, ab105472), MMP2 (Abcam, ab92536) and MMP9 (Abcam, ab137867), ERK1/2 (Cell Signaling, #9126S) or phosphorylated ERK1/2 (Cell Signaling, #4695S) using the indicated antibodies.
4
Immunohistochemical Analysis of Liver Tissues
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The formalin-fixed, paraffin-embedded human and mouse liver tissue sections were subjected to IHC staining. Briefly, the sections were deparaffinized, hydrated, and subjected to antigen retrieval by incubating the slides in a pressure cooker for 15 min in 0.01 M citrate buffer and then incubated with 0.3% hydrogen peroxide (H2O2) in methanol for 10 min to block endogenous peroxidase activity. Then, the sections were incubated with primary antibodies against α-smooth muscle actin (α-SMA) (14395-1-AP, Proteintech, Wuhan, Hubei, China), NLRP3 (19771-1-AP, Proteintech, Wuhan, Hubei, China), GSDMD (20770-1-AP, Proteintech, Wuhan, Hubei, China), IL-1β (16806-1-AP, Proteintech, Wuhan, Hubei, China), S100A8 (ab92331, Abcam, Cambridge, England, UK) or S100A9 (ab63818, Abcam, Cambridge, England, UK) overnight at 4 °C. The cells were washed with PBS and stained with anti-rabbit IHC Secondary Antibody Kit (SP-9001, Zhongshan Golden Bridge, Haidian, Beijing, China). Finally, the sections were visualized with 0.05% 3,3-diamino-benzidine tetrachloride (DAB) until the desired brown reaction product was obtained. The stained sections were observed and photographed under a light microscope (Nikon E400, Chiyoda, Tokyo, Japan).
5
Western Blot Analysis of Inflammation Markers
6
Western Blot Analysis of Liver Proteins
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Total protein was extracted from frozen liver samples by RIPA lysis buffer (Applygen, China) with protease inhibitor. Proteins were determined by the bicinchoninic acid (BCA) method. Samples were centrifuged for 15 min at 4 °C and 12 000 rpm and supernatants were collected. Protein concentrations were measured with a BCA Protein Assay Kit (Pierce). After denaturation, liver protein samples were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore Corp., Billerica, MA, USA). Membranes were blocked for 2 h in 1 × TBST with 5% nonfat milk. Primary antibodies against S100A8 (1:1000; ab92331, Abcam), S100A9 (1:250; ab92507, Abcam), and β-actin (1:1000; ac026, Abclonal) were incubated with the membrane overnight at 4 °C. The membranes were washed three times with TBST. The membrane was then incubated with the corresponding secondary antibodies (HRP goat anti-rabbit IgG; AS014, Abclonal) at room temperature for 1 h and finally were scanned with the Tanon 4200SF fully automated chemiluminescence image analysis system (Shanghai Tianeng Technology Co., ltd., Shanghai, China). The lanes were analyzed by Image J software.
7
Quantification and Western blot analysis
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The concentration of extracted protein was determined using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, China). Equivalent amount of protein was separated using 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After being transferred to 0.2 µm polyvinylidene fluoride membranes, protein bands were blocked with 5% bovine serum albumin for 0.5 h and then incubated with primary antibody: rabbit anti-CD9 antibody (Abcam/ab92726, USA), mouse anti-Hsc70 antibody (Abcam/ab2788), rabbit anti-TSG101 antibody (Abcam/ab125011), rabbit anti-S100A8 antibody (Abcam/ab92331), rabbit anti-S100A9 antibody (Abcam/ab92507), or rabbit anti-S100A12 antibody (Abcam/ab37657) at 4 °C overnight. After that, membranes were incubated with secondary antibodies (Boster, China) conjugated with horseradish peroxidase for 1 h at room temperature and then visualized with a chemiluminescence imaging system (ChemiDoc™ MP Imaging System, Bio-Rad, USA).
8
HepG2 Cells Treated with PO and CCl4
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HepG2 cells were seeded at a density of (4.8 × 105)/mL in 6-well plates and incubated overnight. Then the cells were pretreated with PO (0,1, 2, 2.5, 3, 4 mg/mL) for 24 h, followed by CCl4 (55% saturated solution) intervened for 4 h. HepG2 cells were collected and placed in RIPA lysis buffer (Applygen, China) with protease inhibitor and centrifuged (12 000 rpm for 5 min) to remove impurities at the end of the experiment. Proteins were determined by the bicinchoninic acid (BCA) method. Protein samples (total protein, 50 μg/lane) were separated by 12% polyacrylamide gel electrophoresis and then transferred to membranes (polyvinylidene fluoride, PVDF, Millipore, USA). Membranes were blocked for 1 h in 1 × TBS with 5% nonfat milk. Primary antibodies against S100A8 (1:1000; ab92331, Abcam), S100A9 (1:250; ab92507, Abcam), and β-actin (1:1000; ac026, Abclonal) were incubated with the membrane overnight at 4 °C. The membranes were washed three times with TBST. The membrane was then incubated with the corresponding secondary antibodies (HRP goat anti-rabbit IgG; AS014, Abclonal) at room temperature for 1 h and finally were scanned with the Tanon 4200SF fully automated chemiluminescence image analysis system. The lanes were analyzed by Image J software.
9
Immunohistochemical Analysis of Kidney Pathologies
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Renal tissue was collected from patients undergoing nephrectomy due to kidney stones or renal tumor. Tissue from kidney stone patients was classified as the stone group and normal paracancer tissue from kidney tumor patients was defined as the control group. The samples were fixed with formalin and embedded in paraffin for routine sectioning. Slices of the tissue were incubated with anti-S100A8 antibody (ab92331; Abcam, Cambridge, UK), anti-S100A9 antibody (ab92507; Abcam, Cambridge, UK), anti-uromodulin antibody (A01303-2; Boster, Selangor, Malaysia), anti-OPN (ab8448; Abcam, Cambridge, UK), and anti-albumin antibody (A1363; Abclonal, Wuhan, China) for immunohistochemistry. We used Image J software version 1.52 (National Institute of Mental Health, Bethesda, MD, USA) was used to quantify the relative area of the positive staining area. The ethical review board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology approved the collection and use of tissue samples (2019S1147). The written form of informed consent was obtained from all patients.
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