The colon tissues were homogenized and ultrasonically treated on ice for cell lysis and protein extraction. After determining the protein content, the degeneration process was performed at 100 °C for 5 min. The proteins were separated using 10% SDS-PAGE and transferred to PVDF membranes (0.4-5 μm pore size; Millipore, United States). After blocking with 5% skimmed milk, the membranes were incubated with rabbit anti-TIM3 (1:2000), anti-IL-1β (1:2000), and anti-IL-6 (1:2000) antibodies (Abcam, Cambridge, UK) at 4 °C overnight and then incubated with secondary antibodies labeled with horseradish peroxidase (1:10000, Zhongshan Gold Bridge, Beijing, China) for 2 h at room temperature. The immunoblots were detected using an enhanced chemiluminescent substrate (Millipore) on the ChemiDoc MP system (Bio-Rad, United States). All of the detected protein bands were standardized against β-actin. The semiquantification of each band was performed using ImageJ NIH software (National Institutes of Health, Bethesda, MD, United States).
Anti tim3
Manufactured by Abcam
Sourced in United Kingdom
Anti-TIM3 is a laboratory research antibody that binds to the TIM3 protein. TIM3 is an immune checkpoint receptor that plays a role in regulating T cell function. This antibody can be used to detect and study the TIM3 protein in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 6, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Enhanced chemiluminescent substrate, by Merck Group (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Anti cd56 clone hcd56, by BioLegend (1 mentions)
Anti nkg2d, by Abcam (1 mentions)
Anti granzymeb, by Bioss Antibodies (1 mentions)
Anti pd l1, by Abcam (1 mentions)
Anti hla abc, by Abcam (1 mentions)
Anti ifn γ, by Abcam (1 mentions)
Anti ccr7, by Abcam (1 mentions)
Anti mrp2, by Abcam (1 mentions)
Anti p gp, by Abcam (1 mentions)
Protein loading buffer, by Beyotime (1 mentions)
Nc membrane, by Merck Group (1 mentions)
Anti uqcrc2, by Abcam (1 mentions)
Anti pd 1, by Abcam (1 mentions)
Anti pgc1, by Abcam (1 mentions)
6 protocols using anti tim3
1
Protein Expression Analysis in Colon Tissue
2
Characterization of dNK Cell Phenotype
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Purified human CD3-CD56+ dNK cells from uninfected, infected, and Tim-3-neutralized infected groups were air-dried onto Poly-L-lysine coated slides. After fixation in 4% paraformaldehyde for 30 min, slides were then blocked with goat serum for 1 h at room temperature. dNK cells were incubated overnight at 4°C with anti-Tim-3 (1/200, Abcam), anti-NKG2D (1/200, Abcam), anti-GranzymeA, anti-GranzymeB and anti-Perforin (1/200, all from Proteintech) or for 45 min with anti-CD56 (clone HCD56, Biolegend) and anti-KIR2DL4 (clone mAb33, Biolegend) antibody. After being washed three times with PBS, cells were then incubated with appropriate concentrations of secondary antibodies for 1 h at 37°C. Cy3 rabbit anti-goat IgM (1/500, Bioss) was used as the secondary antibody for anti-Tim-3 antibody, and Cy3 donkey anti-rabbit IgG (1/500, Bioss) was used as secondary antibody for anti-NKG2D, anti-GranzymeA, anti-GranzymeB and anti-Perforin. Subsequently, dNK cells were stained with the DAPI (nucleic acid stain 4’,6-diamidino-2-phenylindole) for 15 min and washed 3 times with PBS. Finally, confocal microscopy of cells was performed using Zeiss LSM880.
3
Immunohistochemical Antibody Panel for Cancer
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The mouse monoclonal antibodies used in this study were: anti-MUC1 (clone BSB-44, Bio SB, dilution 1:50), anti-CEA (clone BSB-31, Bio SB, prediluted), anti-CA19-9 (clone 121SLE, dilution 1:75), anti-MRP2 (clone M2III-6, dilution 1:100), anti-MRP3 (clone M3II-9, dilution 1:5), anti-Pg-P (clone JSB-1, dilution 1:5), anti-TIM3 (clone TIM3/3113, dilution 1:50), anti-PD-L1 (clone ABM4E54, dilution 1:500), anti-HLA-ABC (clone EMR8-5, dilution 1:50), anti-IL-8 (clone 807, dilution 1:50) (Abcam, Cambridge, UK), anti-survivin (clone 8E2, dilution 1:30), anti-TNF-a (clone F6C5, dilution 1:50), and anti-TGF-b (clone TB21, dilution 1:50) (ThermoFisher Scientific, Waltham, MA, USA). The rabbit polyclonal antibodies used were: anti-A2aR (PA-33323, ThermoFisher Scientific, dilution 1:200), anti-IFN-g (ab25101, dilution 1:500), anti-CXCR4 (p-S339) (ab74012, dilution 1:75), anti-CCR7 (ab140758, dilution 14 mg/mL), and anti-IL-6 (ab6672, dilution 1:600) (from Abcam).
4
Western Blot Analysis of Metabolic and Immune Markers
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Cells were collected and lysed with NP40 Lysis Buffer (Beyotime) with 1 mM Phenylmethanesulfonyl fluoride (Beyotime). Each quantity of 106 cells was lysed with 50 μL NP40 buffer. The cell lysates were collected by centrifugation at 13,000 rpm at 4°C for 10 minutes. Protein loading buffer (Beyotime) was added, and the samples boiled at 95°C for 10 minutes. The isolated protein was subjected to SDS-PAGE on 4% to 20% polyacrylamide gels (ACE Biotechnology, Nanjing, China) and transferred to NC membranes (Millipore, St. Louis, MO, USA). The membranes were incubated with a primary antibody and secondary antibody. The primary antibodies included anti-Glut1, anti-LDHA, anti-NRF1, anti-NRF2, anti-GZMB, anti-Actin (Beyotime), anti-SIRT1 (Abclonal, Wuhan, China), anti-HK2, anti-CPT1α, anti-PGC1, anti-MTCO2, anti-TFAM, anti-GZMA, anti-GZMK, anti-PRF1, anti-PD-1, anti-TIM-3, anti-CTLA-4, anti-NDUFB8, anti-SDHB, anti-MTCO1, anti-UQCRC2, and anti-ATP5A (Abcam, Cambridge, UK). The secondary antibodies included HRP-labeled goat anti-rabbit and HRP-labeled goat anti-mouse antibodies (Beyotime). Finally, the proteins were detected by West Femto Maximum Sensitivity Substrate (ThermoFisher).
5
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Total protein was isolated using radioimmunoprecipitation assay (RIPA) lysis buffer that contained a protease and phosphatase inhibitor cocktail (Solarbio, Beijing, China). The protein extracts were separated through SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes ((Millipore, Billerica, MA, USA). The membranes were subsequently blocked in 5% defatted milk or bovine serum albumin (for phosphorylated protein), and incubated with rabbit anti-human antibodies overnight at 4°C. The primary antibodies used were anti-β-actin (1:1000; Bioss, Beijing, China), anti-TIM-3 (1:1000; Abcam), anti-SMAD7 (1:1000; Proteintech, Rosemont, IL, USA), anti-SNAIL1 (1:800; Proteintech), anti-E-cadherin (1:4000; Proteintech), anti-N-cadherin (1:4000; Proteintech), anti–Vimentin (1:4000; Proteintech), anti-MMP2 (1:1000; Proteintech), anti-MMP9 (1:1000; Proteintech), and anti-SMAD2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-SMAD2 (Ser 465/467, 1:1000; Cell Signaling Technology). The secondary antibody used was horseradish peroxidase-conjugated goat anti-rabbit antibody (1:10,000; Abcam). Proteins were visualized with an enhanced chemiluminescence (ECL) detection system (Meilun). Images were captured using the Odyssey Fc Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
6
Synovial Tissue Immunohistochemistry for Inflammation
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The synovial tissue was immersed in 4% paraformaldehyde, and then it was dehydrated, embedded in para n and then cut into 5µm thickness. The sections were placed in EDTA for antigen repair. Then, they were blocked with goat serum for 1 h at room temperature. After the blocking procedure was nished, the primary antibodies were added and the sections were incubated at 4 °C for 24h. The primary antibodies used in the process included anti-Tim-3 (1:200, Abcam, UK), anti-Gal-9 (1:500, Abcam, UK), anti-CD86 (1:500, Abcam, UK) and anti-CD206 (1:500, Abcam, UK). The sections were washed with PBS for three times, and then they were incubated with secondary antibody at 37 °C for 30 min. Then, the nuclei were counter-stained with DAPI. The images were visualized with uorescence microscope (Nikon Eclipse C1, Japan).
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