Cells were incubated in 24-well plates with coverslips at 37°C for 24 h. Specimens were stained with the primary antibody for 60 min at 4°C and then stained with an FITC-conjugated secondary antibody (Proteintech, Rosemont, IL, USA) and DAPI (Beyotime Institute of Biotechnology, Shanghai, P. R. China). The primary mAbs used for staining included those for B7-H3 (Cell Signaling Technology, CST, Danvers, MA, USA; D9M2L), CD70 (Abcam, Cambridge, MA, USA; ab175389), TIM-3 (Abcam; ab47997), VISTA (CST; D5L5T), B7-H3 mAb (J42), and J42-scFv-Fc. The images were captured using confocal microscopy. For RNA-seq and survival analysis, the data were downloaded from were downloaded from the Gene Expression Profiling Interactive Analysis (GE-PIA) (http://gepia.cancer-pku.cn/ ), which has access to The Cancer Genome Atlas (TCGA).
Tim 3
Manufactured by Abcam
Sourced in United Kingdom
TIM-3 is a cell surface receptor that plays a role in the regulation of immune responses. It is expressed on various immune cell types, including T cells, natural killer cells, and dendritic cells. TIM-3 functions as an inhibitory receptor, and its interaction with its ligands can modulate immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated secondary antibody, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Xylene, by Thermo Fisher Scientific (1 mentions)
Iba 1, by Abcam (1 mentions)
Donkey anti mouse igg, by Abcam (1 mentions)
Gal 9, by Abcam (1 mentions)
Casp1, by Santa Cruz Biotechnology (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Goat anti rabbit igg, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Silanized slides, by Agilent Technologies (1 mentions)
Ctla4, by Abcam (1 mentions)
Acetone pa, by Merck Group (1 mentions)
Stat1, by Abcam (1 mentions)
5 protocols using tim 3
1
Immunofluorescence Analysis of Immune Checkpoint Proteins
2
Immunohistochemical Analysis of Glioma Markers
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All glioma patient tissues, including grade 2, 3, and 4, used in IHC were obtained surgically, and the number of patients and pathological information were recorded in Supplementary Table S1 . Formalin-fixed, paraffin-embedded glioma blocks were serially sectioned at 4 µm thickness and deparaffinized in xylene (Thermo Fisher Scientific). The sections were immersed in ethanol, rehydrated, and incubated with goat serum to block nonspecific binding. They were then incubated at 4 °C overnight with primary antibodies against Gal-9 (mouse monoclonal Ab; Abcam, Cambridge, UK), TIM-3 (rabbit monoclonal Ab; Abcam), GFAP (rabbit monoclonal Ab; Abcam), Iba-1 (rabbit monoclonal Ab; Abcam), NLRC4 (rabbit polyclonal Ab; Abcam), or caspase-1 (mouse monoclonal Ab; CASP-1; Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies, including goat anti-rabbit IgG (Abcam) and donkey anti-mouse IgG (Abcam), were applied at room temperature for 1 h. The sections were examined under a confocal laser scanning microscope (Zeiss LSM; Carl Zeiss AG, Jena, Germany) and quantified using the ImageJ software v1.52a (NIH, Bethesda, MD, USA).
3
Western Blot Analysis of GAPDH and TIM-3
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Total protein was extracted, and BCA method was carried out to test protein concentration. Then, protein was separated on 10% SDS‐PAGE. After finishing electrophoresis, protein was transferred to PVDF membrane. Then, the membrane was subjected to 5% skim milk. The membranes were processed with primary antibodies against GAPDH and TIM‐3 (Abcam). The membrane was incubated with horseradish‐peroxidase‐bound secondary antibodies for 2 h. Then, the membrane was visualized by an ECL system.
4
Immunohistochemical Analysis of Splenic Tissue
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The splenic tissue was cryosectioned at a thickness of 5 μm, and the sections were placed on silanized slides (Dako, Carpinteria, CA, USA) and fixed with acetone P.A. (Merck) for 10 minutes. For the inhibition of non-specific binding, the sections were incubated in a solution containing 0.4% BSA for 20 minutes at room temperature. The excess blocking solution was discarded, and the primary antibodies for the detection of CD21 (Bio-Rad AbD Serotec), CTLA-4 and TIM-3 (Abcam) were added for 18 hours at 4 °C. The reaction was revealed by a secondary anti-mouse IgG antibody conjugated to phycoerythrin (PE-red) and an anti-rabbit IgG antibody conjugated to fluorescein isothiocyanate (FITC). The incubation with the secondary antibodies was performed in a dark room for 30 minutes at room temperature. The slides were assembled using Fluoromount-g medium containing DAPI (4′, 6-diamino-2-phenylindole) (Thermo Fisher Scientific), and the reaction was observed under a fluorescence microscope. The images were processed and overlaid with ImageJ software (NIH, USA).
5
Synovial Tissue Protein Analysis
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Protein samples were obtained from the synovial tissue and the protein concentration was determined. SDS-PAGE method was used to separate the protein samples, then they were transferred to PVDF membrane and blocked with TBS containing 0.05%-20 (TBST) and 5% non-fat milk powder for 2 hours. The specimens were incubated with primary antibodies including Tim-3 (1:1000, Abcam, UK) and STAT1
(1:1000, Abcam, UK) at 4 °C for 24h. The membranes was washed with TBST for 3 times and incubated with secondary antibody for 2 hours at room temperature. Images were captured with a chemiluminescence imaging system (ChemiScope 6100, Shanghai, China).
(1:1000, Abcam, UK) at 4 °C for 24h. The membranes was washed with TBST for 3 times and incubated with secondary antibody for 2 hours at room temperature. Images were captured with a chemiluminescence imaging system (ChemiScope 6100, Shanghai, China).
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