The following reagents were acquired from Sigma-Aldrich (St. Louis, MO, USA): AFB1, ionomycin, PMA, and RPMI-1640 medium. The fluorescently labeled antibodies targeting IFN-γ, STAT1, T-bet, IL-9, IRF4, IL-17A, IL-21, RORγT, STAT3, IL-22, AhR, TNF-α, IL-10, TGF-β1, FoxP3, and the buffers for red blood cell permeabilization/fixation were procured from BioLegend (San Diego, CA, USA). The Golgi-Plug and RORγT reagents were acquired from BD Biosciences (San Diego, CA, USA). The primary antibodies used in this study, including IFN-γ, T-bet, IL-9, IL-17A, RORγT, TNF-α, IL-10, and FoxP3, were acquired from Santa Cruz Biotechnology (Dallas, TX, USA). The FcR blocking reagent was acquired from Miltenyi Biotech (Bergisch Gladbach, Germany). The nitrocellulose membranes utilized in this study were acquired from Bio-Rad Laboratories (Hercules, CA, USA). The primers utilized in this work were acquired from GenScript (Piscataway, NJ, USA). The Merck, Darmstadt, Germany’s chemiluminescence kit was utilized to conduct Western blotting. The TRIzol® reagent used in this study was obtained from Life Technologies (Carlsbad, CA, USA). The SYBR® Green and cDNA kits utilized in this study were purchased from Applied Biosystems (Foster City, CA, USA).
Ifn γ
Manufactured by Santa Cruz Biotechnology
Sourced in United States
IFN-γ is a cytokine involved in immune response. It is produced by various cells, including T cells and natural killer cells. IFN-γ plays a role in the activation and regulation of the immune system.
Automatically generated - may contain errors
Lab products found in correlation
Il 13, by Santa Cruz Biotechnology (5 mentions)
Interleukin 6 (il 6), by Santa Cruz Biotechnology (4 mentions)
Il 1β, by Santa Cruz Biotechnology (3 mentions)
Interleukin 4 (il 4), by Santa Cruz Biotechnology (3 mentions)
Tnf α, by MyBioSource (3 mentions)
Tnf α, by Abcam (3 mentions)
Il 10, by Santa Cruz Biotechnology (2 mentions)
Tnf α, by Santa Cruz Biotechnology (2 mentions)
Biotinylated secondary antibody, by Agilent Technologies (2 mentions)
Il 12p40, by Santa Cruz Biotechnology (2 mentions)
Axio scope a1, by Zeiss (2 mentions)
Universal quick kit, by Vector Laboratories (2 mentions)
Chemiluminescence kit, by Merck Group (1 mentions)
Tgf β1, by BioLegend (1 mentions)
Rorγt, by BD (1 mentions)
Il 10, by BioLegend (1 mentions)
T bet, by Santa Cruz Biotechnology (1 mentions)
Il 17a, by Santa Cruz Biotechnology (1 mentions)
Rorγt, by Santa Cruz Biotechnology (1 mentions)
Sybr green and cdna kits, by Thermo Fisher Scientific (1 mentions)
13 protocols using ifn γ
1
Multiparametric Analysis of Immune Cell Activation
2
Retinal Protein Profiling via Western Blotting
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Retinal and cellular protein were harvested and homogenized in lysis buffer (RIPA, Biocolors, Shanghai, China) containing protease and phosphatase inhibitor mini tablets (Thermo Fisher Scientific, no. 88668; Waltham, MA, USA). The protein concentration was determined with bicinchoninic acid assay. Western blotting was performed as described previously. Briefly, 30 μg of protein was loaded per lane on a 10% SDS-PAGE. The membrane blots were saturated with 5% BSA in PBST for 1 h at room temperature and then incubated overnight at 4 °C with antibodies against TNF-α, RIP3 and MLKL (1 : 1000, Abcam), CCL2 (1 : 1000, Novus Biologicals), RIP1 (1 : 1000, BD Bio-Sciences), IL-6 (1 : 1000, R&D Systems, Minneapolis, MN, USA), IL-1β, IL-23, IFN-γ (1 : 500, Santa Cruz, CA, USA) and β-actin (1:2000, Abcam) were used. For the assessment of RIP1 phosphorylation, lysates were first immunoprecipitated with anti-RIP1 antibody overnight. The immunocomplexes were then analyzed by western blotting with anti-RIP1 antibody and anti-phosphotyrosine antibody (p-Ser, 1 : 200, Santa Cruz). The gray intensity of proteins was measured using Image J software (US National Institutes of Health).
3
Microglial Polarization Dynamics in Alzheimer's
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To observe microglial polarization, we used typical stimuli: LPS and IFN-γ for M1 polarization, and anti-inflammatory cytokines (IL-4 and IL-13) for M2 polarization [4 (link), 16 (link)]. The microglia were divided into six groups: the control group (Con), the LPS/IFN-γ treatment group (LPS/IFN-γ), the IL-4/IL-13 treatment group (IL-4/IL-13), the Aβ1-24 treatment group (Aβ), the LPS/IFN-γ and Aβ1-24 co-treatment group (LPS/IFN-γ+Aβ) and the IL-4/IL-13 and Aβ1-24 co-treatment group (IL-4/IL-13+Aβ). Microglia in the LPS/IFN-γ and LPS/IFN-γ+Aβ groups were incubated with LPS (100 ng/mL, Sigma) and IFN-γ (100 U/mL, Santa Cruz Biotechnology) for 24 h. For the IL-4/IL-13 and IL-4/IL-13+Aβ groups, the microglia were incubated with IL-4 (20 ng/mL, Santa Cruz Biotechnology) and IL-13 (20 ng/mL, Santa Cruz Biotechnology) for 24 h. For the LPS/IFN-γ+Aβ and IL-4/IL-13+Aβ groups, the microglia were pre-incubated with Aβ1-24 (1 ng/mL, Sigma) for 2 h before LPS/IFN-γ or IL-4/IL-13 were added, and then the microglia were incubated with both Aβ and these inflammatory substances for 24 h.
4
Immunohistochemical Analysis of Cytokine Expression
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IHC analysis was conducted as previously described [28 ]. Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min to remove endogenous peroxidase. Antigen retrieval was carried out with sodium citrate buffer (0.1 M) using the microwave method. The slides were incubated with normal serum to block nonspecific binding and then incubated for 1 h with primary antibodies (diluted 1:100 to 1:200) such as IFN-γ (sc-74104, Santa Cruz Biotechnology, Dallas, TX, USA), IL-12p40 (sc-57258), IL-4 (sc-73318), IL-13 (sc-1776), TNF-α (MyBioSource, San Diego, CA, USA), and IL-6 (Novus Biologicals, Littleton, CO, USA). The slides were incubated for 10 min with biotinylated secondary antibodies (Vector Laboratories, PK-7800, Burlingame, CA, USA) and horseradish peroxidase-conjugated streptavidin. Signals were detected using the 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution, and the cells were counterstained with Mayer’s hematoxylin.
5
Isolation and Analysis of CD4+ T Cells
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Peripheral blood was collected into heparinized tubes, diluted to 1.5 ml final volume with PBS, and stored at 4°C for <4 h. Samples were layered onto Ficoll Paque Plus, and peripheral blood mononuclear cells were isolated by using a density gradient separation technique. An automated magnetic bead–based positive selection protocol was used to isolate CD4+ T cells (Stemcell Technologies, Vancouver, BC, Canada). All Western blot analyses were then performed according to standardized protocols above described. Here, primary Abs used were as follows: IL-17 (1:250; Santa Cruz Biotechnology) and IFN-γ (1:250; Santa Cruz Biotechnology). Representative blot images from 3 separate analyses are given.
6
Immunohistochemical Analysis of Inflammatory Cytokines
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Immunohistochemical analysis was performed according to our previous study14 (link). Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min, to remove endogenous peroxidase. Antigen retrieval was performed with the sodium citrate buffer (0.1 M), using the boiling method. The slides were incubated with normal serum to prevent nonspecific binding and then incubated overnight at 4 °C with the following primary antibodies (diluted 1:100 or 1:200): IFN-γ (Santa Cruz, sc-74104), TNF-α (MY BioSource, CA, USA, MBS175453), IL-1β (Santa Cruz, sc-1251), IL-6 (Santa Cruz, sc-7920), IL-10 (Santa Cruz, sc-73309), IL-13 (Santa Cruz, sc-1776), and IL-17 (Abcam, MA, USA, ab79056). The slides were incubated for 2 h with biotinylated secondary antibody (1:500; DAKO, Carpinteria, CA, USA) and horseradish-peroxidase conjugated streptavidin. Signals were detected using the 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution, and cells were counterstained with Mayer’s hematoxylin.
7
Cytokine Expression in Murine Spleen
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At 21 and 42 days of age, splenic samples of eight mice in each group were taken to determine the cytokine protein expression levels by western blot.
The spleen was ground into homogenate and then proteins were extracted with RIPA lysis buffer and kept in laemmli loading buffer. Protein samples were resolved on SDS-PAGE (10%–15% gels) and transferred to nitrocellulose filter membranes. Membranes were blocked with 5% fat-free milk for 1h and incubated with primary antibodies overnight at 4°C. The primary antibodies were TNF-α, IFN-γ, TGF-β, and IL-2, IL-10 (Santa Cruz, USA). The membranes were then washed with PBS-tween and incubated with biotin-conjugated secondary antibodies (Santa cruz, USA) for 1h, and washed again with PBS-tween. Blots were visualized by ECLTM (Bio-Rad, Hercules, CA, USA) and X-ray film.
The spleen was ground into homogenate and then proteins were extracted with RIPA lysis buffer and kept in laemmli loading buffer. Protein samples were resolved on SDS-PAGE (10%–15% gels) and transferred to nitrocellulose filter membranes. Membranes were blocked with 5% fat-free milk for 1h and incubated with primary antibodies overnight at 4°C. The primary antibodies were TNF-α, IFN-γ, TGF-β, and IL-2, IL-10 (Santa Cruz, USA). The membranes were then washed with PBS-tween and incubated with biotin-conjugated secondary antibodies (Santa cruz, USA) for 1h, and washed again with PBS-tween. Blots were visualized by ECLTM (Bio-Rad, Hercules, CA, USA) and X-ray film.
8
Immunohistochemical Analysis of Inflammatory Cytokines
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Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min to remove endogenous peroxidase. Antigen retrieval was carried out with sodium citrate buffer (0.1 M) using the boiling method. The slides were incubated with normal serum to block nonspecific binding and then incubated overnight at 4°C with the following primary antibodies (diluted 1:100 or 1:200): IL-1β (Santa Cruz, CA, USA, sc-1251), IL-6 (Santa Cruz, sc-7920), IL-13 (Santa Cruz, sc-1776), IFN-γ (Santa Cruz, sc-74104) and TNF-α (MY BioSource, CA, USA, MBS175453). The slides were incubated for 2 h with biotinylated secondary antibody (1:500; DAKO, Carpinteria, CA, USA) and horseradish-peroxidase conjugated streptavidin. Signals were detected using 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution, and cells were counterstained with Mayer’s hematoxylin.
9
Immunohistochemistry and Immunofluorescence for Tight Junction Proteins
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For immunohistochemistry staining, the sections were boiled 10 min in 10 mmol/L citrate (pH 6.0) for antigen retrieval. The slides were then incubated with mouse monoclonal antibodies against claudin-1, occludin (Santa Cruz Biotechnology, Dallas, TX, United States), TNF-α, and IL-17 (Sigma), and rabbit polyclonal antibodies against zo-1 (1:150; Invitrogen, Carlsbad, CA, United States) and IFN-γ (1:300; Santa Cruz Biotechnology), followed by peroxidase-conjugated secondary antibodies (1:1500). The signals were visualized by a diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA, United States). For immunofluorescence staining, the sections were incubated with mouse monoclonal antibodies against claudin-1 and occludin (1:200; Santa Cruz Biotechnology) and rabbit polyclonal antibody against zo-1 (1:150; Invitrogen), and subsequently with FITC- or Cy3-conjugated secondary antibodies. Images were captured under a Leica DMIRE2 confocal laser scanning microscope.
10
Comprehensive Protein Expression Analysis
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Standard immunoblotting and immunehistochemistry protocols were carried out. Antibodies used were AR (N-20, Santa Cruz, CA, USA). PTen, AKT, p-Akt, NFκβ, IFNγ, HIF1α and STAT3 were obtained from Santa Cruz. CD3 was from DAKO (Santa Clara, CA USA) and CD45R was from Biolegend (San Diego, CA, USA). The Vectastain avidin–biotin complex (Vector Labs, Peterborough, U.K.) was used for detection, using diaminobenzidine chromogenic substrate. Negative controls were included lacking primary antibody. Images were captured using a Leica DM1000 microscope.
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