Fresh tissues were formalin-fixed immediately followed surgical resection and were then embedded in paraffin. Tissues were processed as previously described57 (link). Briefly, fixed tissues were then slide mounted, deparaffinized using xylene and ethanol, and then re-fixed in formalin for 15 min followed by antigen retrieval. Slides were stained with the following antibodies: CD20 (Clone L26, ThermoFisher, 1:100, Cat# MA5-13141), CD4 (Clone D7D27, Cell Signaling, 1:100, Cat# 25229), CXCR5 (Clone D6L36, Cell signaling, 1:100, Cat# 72172), Tbet (Clone 4B10, Abcam, 1:100, Cat# ab91109). Quantification of cells and TLS were performed by a HNSCC pathologist. Specifics of these quantifications are outlined in Fig. Legends and definitions of a TLS were consistent across three independent pathologists.
T bet
Manufactured by Abcam
Sourced in Hong Kong
T-bet is a transcription factor that plays a critical role in the development and function of T helper 1 (Th1) cells. It is involved in the regulation of genes associated with Th1 cell differentiation and promotes the expression of interferon-gamma (IFN-γ).
Automatically generated - may contain errors
Lab products found in correlation
Gata3, by Cell Signaling Technology (2 mentions)
Tia 1, by Beckman Coulter (1 mentions)
Granzyme b, by Agilent Technologies (1 mentions)
P stat6, by Abcam (1 mentions)
Histamine dihydrochloride, by Merck Group (1 mentions)
Apigenin, by Merck Group (1 mentions)
Nf κb, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Abcam (1 mentions)
Socs1, by Abcam (1 mentions)
Total rna extraction kit, by MP Biomedicals (1 mentions)
Pd l1, by R&D Systems (1 mentions)
Envision system hrp, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Agilent Technologies (1 mentions)
P stat4, by Abcam (1 mentions)
P stat1, by Abcam (1 mentions)
Stat4, by Abcam (1 mentions)
C maf, by Abcam (1 mentions)
Goat anti rabbit igg hrp antibody, by Santa Cruz Biotechnology (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Ripa buffer, by Abcam (1 mentions)
8 protocols using t bet
1
Immunohistochemical Profiling of Tumor-Infiltrating Lymphocytes
2
Immunohistochemical Evaluation of T-cell Markers
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The antibodies used for IHC were CD3 (M7254, DAKO), CD4 (790–4423, VENTANA), CD8 (M7103, DAKO), CD30 (M0751, DAKO), TIA‐1 (IM2550, BECKMAN COULTER), Granzyme B (M7235, DAKO), T‐bet (4B10, Abcam), CXCR3 (1C6, BD), GATA3 (5852, Cell signaling Technology), and CCR4 (1G1, BD). Except for T‐bet, CXCR3, GATA3, and CCR4, the cutoff value for positivity was 30%. If neoplastic cells were positive for TIA‐1 and/or Granzyme B, the patient was considered positive for cytotoxic molecules.
3
Apigenin Modulates Allergic Responses
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Apigenin (purity ≥95%), OVA (grade V), aluminum hydroxide, and histamine
dihydrochloride were purchased from Sigma-Aldrich Co, St Louis, Missouri.
Montelukast was obtained from Cipla Limited, Mumbai, India. Mouse OVA-specific
IgE, total IgE, total IgG1, β-hexosaminidase, IL-4, IL-5, IL-13, IL-17, IFN-γ,
and Leukotriene C4 enzyme-linked immunosorbent assay (ELISA) kit were obtained
from Bethyl Laboratories Inc, Montgomery, Texas. The primary antibodies of
GATA3, T-bet, NF-κB, IκBα, p-STAT6, suppressor of cytokine signaling 1 (SOCS1),
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Abcam,
Cambridge, Massachusetts. Total RNA extraction kit and real time-polymerase
chain reaction (RT-PCR) kit were purchased from MP Biomedicals India Private
Limited, India.
dihydrochloride were purchased from Sigma-Aldrich Co, St Louis, Missouri.
Montelukast was obtained from Cipla Limited, Mumbai, India. Mouse OVA-specific
IgE, total IgE, total IgG1, β-hexosaminidase, IL-4, IL-5, IL-13, IL-17, IFN-γ,
and Leukotriene C4 enzyme-linked immunosorbent assay (ELISA) kit were obtained
from Bethyl Laboratories Inc, Montgomery, Texas. The primary antibodies of
GATA3, T-bet, NF-κB, IκBα, p-STAT6, suppressor of cytokine signaling 1 (SOCS1),
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Abcam,
Cambridge, Massachusetts. Total RNA extraction kit and real time-polymerase
chain reaction (RT-PCR) kit were purchased from MP Biomedicals India Private
Limited, India.
4
Immunohistochemical Profiling of Immune Markers
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IHC was performed on formalin-fixed, paraffin-embedded tissue sections. Serial sections (5-μm thick) were dewaxed and rehydrated. Heat-induced antigen retrieval was followed by endogenous peroxidase activity blockade with hydrogen peroxide. The sections were incubated overnight at 4°C with the following primary antibodies: CD207 (1:50; Atlas Antibodies), VE1 (1:50; Spring Bioscience), FOXP3 (1:75; Abcam), and PDL1 (1:50; R&D Systems). For antigen visualization, a peroxidase-labeled secondary antibody (EnVision/HRP system; DAKO) was used. Subsequently, the sections were rinsed in the kit buffer and immersed in diaminobenzidine stain. The expression of GATA3 (1:100; Cell Signaling Technology) and T-bet (1:50; Abcam) was performed via double IHC staining using DouMaxVision immunohistochemical double dye kits (KIT-9998; Maixin Biotech) according to the manufacturer's instructions, with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as the blue-black chromogen for alkaline phosphatase and 3-amino-9-ethylcarbazole as the red chromogen for horseradish peroxidase. The sections were counterstained with Harris' hematoxylin and then mounted. For the negative controls, phosphate-buffered saline was used instead of primary antibody.
5
Immunohistochemical Staining of Tissue Sections
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Paraffin-embedded specimens were cut into 4 μm thick sections. The slides were dewaxed by heating at 60 °C for 60 min followed by deparaffinizationin xylene and rehydration in graded alcohol. The slides were then put in Citrate Buffer solution (pH 6.0) and microwaved for 10 min at low power for antigen retrieval. Deparaffinized sections were stained with the following antibodies: CD8 1:150 (Abcam ab17147), CD4 1:250 (Abcam ab846), T-bet 1:100 (Abcam ab91109), CD68 1:100 (Dako M0814), CD57 1:150 (Abcam ab82749), Isotype Control (Abcam ab91353). The slides were then incubated in 3% H2O2 for 20 min for removal of endogenous peroxidase activity and subsequently incubated with secondary antibody (DAB) at 37 °C for 30 min. The tissue sections were immersed in a solution of 3, 3′-diaminobenzidine tetrahydrochloride (Dako, Hamburg, Germany) and then counterstained with hematoxylin.
6
Western Blot Analysis of Transcription Factors
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Total protein was isolated from PBMCs by using RIPA buffer (Abcam, Cambridge, United Kingdom). The amount of proteins was quantified by BCA Protein assay kit (Thermo Fisher Scientific, Rockford, IL, United States), with bovine serum albumin (BSA) as a standard. Each protein sample was loaded onto 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Schleicher & Schuell BioScience, Germany) for 90 min at 4°C, and blocked with 5% skim milk in TBST (1 M Tris–HCl, 5 M NaCl, 10% Tween-20) for 1 h at room temperature. The blots were incubated with anti-GATA3, -T-bet, -C-maf, -STAT1, -P-STAT1, -STAT4, -P-STAT4 or -beta-actin (all from Abcam) antibodies overnight at 4°C. Primary antibody-bound membranes were incubated with goat anti-rabbit IgG-HRP antibody (Santa Cruz Biotechnology, United States) for 1 h at room temperature. The target protein was visualized with enhanced chemiluminescence system (GE Healthcare Life Sciences, United States), followed by analysis using ChemiDoc XRS (Bio-Rad).
7
Protein Expression Analysis in Lung Tissue
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The right lung was removed, and total protein was extracted using Total Extraction Sample Kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocols. Protein concentrations were determined using the BCA method (cat. no. 23225; Thermo Fisher Scientific, Inc.). Proteins (30 µg) were resolved by 10% SDS-PAGE, and then transferred to a PVDF membrane. Following blocking with TBST supplemented with 5% skim milk powder for 2 h at room temperature, the PVDF membranes were incubated with antibodies against GATA3 (1:1,000; cat. no. ab106625; Abcam), STAT6 (1:2,000; cat. no. ab32520; Abcam), T-bet (1:500; cat. no. ab91109; Abcam) or Runx3 (1:1,000; cat. no. ab135248; Abcam) at 4°C overnight. The membranes were subsequently incubated with HRP-conjugated goat anti-mouse secondary antibody (1:5,000; cat. no. ab205719; Abcam) the following day for 1 h at room temperature. Signals were visualized using ECL Chemiluminescence Detection kit (cat. no. SW2010; Beijing Solarbio Science & Technology Co., Ltd.). Protein expression levels were semi-quantified using Gel-Pro-Analyzer software (version 4.0; Media Cybernetics, Inc.) with β-actin as the loading control.
8
Immunoblotting Analysis of T-Cell Signaling Pathways
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T‐cells were lysed in RIPA Lysis buffer and protein concentrations were determined based on BCA assay (Thermo Fisher Scientific, USA). PAGE‐separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Germany) by wet transfer method. Membranes were incubated with the antibodies against HK2 (Santa Cruz, USA, Cat sc‐130358, 1:500), PFKFB4 (Abcam, Hong Kong, Cat ab137785, 1:2000), T‐bet (Abcam, Hong Kong, Cat ab181400, 1:1000), RORg (Thermo Fisher Scientific, USA, Cat 14‐6988‐82, 1:200), p‐AKT (Ser473, Cat 4060S, 1:2000), p‐S6 (S235/236, Cat 4858S, 1:2000), phosphorylation of mTOR (Ser2448, Cat 5536T, 1:2000), mTOR (Cat 2983T, 1:2000), Raptor (Cat 2280T, 1:2000) and β‐actin (Cat 4967S, 1:2000) (all from Cell Signalling Technology, USA) at 4°C overnight. Primary antibodies were identified with horseradish peroxidase‐labelled anti‐rabbit (Cell Signalling Technology, USA, Cat 7074S, 1:2000) or anti‐mouse (Cell Signalling Technology, USA, Cat 7076S, 1:2000) secondary antibodies. Signals were visualized with a Chemilucent Plus Western Blot Enhancing Kit (Millipore, Germany).
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