Prostatic sections were fixed overnight in a 10% buffered paraformaldehyde solution. The tissues were ethanol-dehydrated and embedded in paraffin wax. Sections of 5 µm were cut with microtome, deparaffinized at 70°C for 20 min, incubated 5 minutes each in xylene, ethanol-xylene, and 100% ethanol. Finally, the samples were washed with distilled water to be processed for immunohistochemical analysis. Immunolabeling was done by using Mouse/Rabbit PolyDetector Plus HRP/DAB (BSB 0259; BioSB) Kit according to the manufacturer's instructions. The antibodies used were TNF-α (50 µg/mL) (Abcam 1793; Massachusetts, USA), IFN-γ (50 µg/mL) (Abcam 7740; Massachusetts, USA), CD45 (1 µg/mL) (Abcam 10558, Massachusetts, USA), IL-17 (1 µg/mL) (Abcam 79056, Massachusetts, USA), IL-6 (74 µg/mL) (Abcam 6672; Massachusetts, USA), IL-4 (0.5 µg/Ml) (Abcam 11524; Massachusetts, USA), and PCNA (100 µg/mL) (Santa Cruz-53407, Texas, USA). After the immunohistochemistry procedure, microphotographs were taken and the relative expression percentages were semiquantified in the Image-Pro Premier software 9.1.
Interleukin 4 (il 4)
Manufactured by Abcam
Sourced in United States, United Kingdom
IL-4 is a cytokine that plays a key role in the regulation of immune and inflammatory responses. It is primarily produced by activated T cells, mast cells, and basophils. IL-4 is involved in the differentiation of naive T cells into Th2 cells and the promotion of IgE class switching in B cells.
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Lab products found in correlation
Ifn γ, by Abcam (9 mentions)
Il 10, by Abcam (6 mentions)
Tnf α, by Abcam (5 mentions)
Il 13, by Abcam (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Gapdh, by Abcam (3 mentions)
Lipopolysaccharides (lps), by Abcam (3 mentions)
Il 17, by Abcam (2 mentions)
Interleukin 6 (il 6), by Abcam (2 mentions)
Elisas for il 2, by Abcam (2 mentions)
Prism 4, by GraphPad (2 mentions)
Ifn γ, by BioLegend (2 mentions)
Iba 1, by Abcam (2 mentions)
Gat 3, by Abcam (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Hmgb1, by Abcam (2 mentions)
Lipopolysaccharides (lps), by Corning (2 mentions)
Interleukin 4 (il 4), by Corning (2 mentions)
Transwell plate, by Corning (2 mentions)
32 protocols using interleukin 4 (il 4)
1
Immunohistochemical Analysis of Prostatic Tissues
2
Multiplex Biomarker Profiling in Plasma
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A panel of 26 biomarkers was measured in all plasma samples. Concentrations of HMGB-1, IL-1β, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-17-A, nucleosomes, Reg3A, ST2, TGF-β1 and TNFα were measured using ELISA kits (all from R&D Systems, Minneapolis, MN, except for HMGB-1 [MyBioSource, San Diego, CA], IL-4 [Abcam, Cambridge, UK] and nucleosomes [Roche, Indianapolis, IN]), and concentrations of CD40L, gp130, IFN-γ, IL-2Rα, IL-6Rα, IL-15, IL-22, IL-23, E-selectin, syndecan-1, thrombomodulin and TNFRI were measured using customized, magnetic bead-based, multi- or monoplex assays (R&D Systems). The magnetic beads were analysed on a Luminex LX-200 instrument (R&D Systems).
3
Protein Expression Analysis in Cells
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Cells were lysed in RIPA buffer (Beyotime, Nantong, China) supplemented with phenylmethylsulfonyl fluoride (Beyotime, Nantong, China). Proteins were then separated by 15% SDS-PAGE gel and transferred onto the PVDF membrane (Millipore, Billerica, MA, USA). β-actin (Abcam, Cambridge, MA, USA), JAK2 (Abcam, Cambridge, MA, USA), STAT1 (Abcam, Cambridge, MA, USA), IFN-γ (Abcam, Cambridge, MA, USA), IL-4 (Abcam, Cambridge, MA, USA), S100A4 (Abcam, Cambridge, MA, USA), p-JAK2 (Abcam, Cambridge, MA, USA) and p-STAT1 (Abcam, Cambridge, MA, USA) primary antibodies and fluorescent dye-labeled secondary antibody were used. The density of protein bands was measured by an infrared imaging system (LI-COR, Lincoln, NE, USA).
4
Cytokine Profile Analysis in Rats
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Peripheral blood samples were collected from rats killed by decapitation at 3 and 8 weeks after immunization (n = 3 per time point/group). ELISAs for IL-2, IL-4, TNF-α (all from Abcam, Cambridge, MA), and IFN-γ (BioLegend Inc., San Diego, CA) were performed as previously described40 (link)41 (link)42 (link). Optical density was measured at 450 nm and analyzed using GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA).
5
Immunohistochemical Profiling of Tumor Samples
6
Placental Cytokine Expression Analysis
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The placental tissues of both Groups were fixed in 10% buffered formalin and embedded in paraffin. A tissue sections of 5µ thickness was processed for immunohistochemistry of pro‐inflammatory (IL‐6, TNF‐α) and anti‐inflammatory (IL‐4, IL‐10) cytokines. For the antigen retrieval, Tris EDTA (pH‐9.0) buffer was used. The sections were incubated overnight in humid chamber at 4°C with primary antibodies IL‐6 (dilution 1:200, rabbit polyclonal, Abcam Inc, Cambridge, UK), TNF‐α (dilution 1:150, rabbit polyclonal, Abcam Inc), IL‐4 (dilution 1:200, rabbit polyclonal, Abcam Inc), and IL‐10 (dilution 1:150, rabbit polyclonal, Abcam Inc). Polymer based Envision plus Kit (Dako Cytomation, Glostrup, Denmark) was used for secondary antibody and chromogenic visualization reaction. For the negative control, primary antibody was replaced with immunoglobulin G, isotype‐specific.
7
Cerebellum Protein Expression Analysis
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The animals were sacrificed by decapitation and the cerebellums were dissected and homogenized in 66 mM Tris–HCl (pH 7.4), 1 % SDS, 1 mM EGTA, 10 % glycerol, 1 mM sodium ortho-vanadate, and 1 mM sodium fluoride containing protease inhibitor cocktail (Roche, Mannheim, Germany). Samples were subjected to electrophoresis and immunoblotting as in Felipo et al. [42 (link)]. Primary antibodies were against IL-4, IL-10, iba-1, Ym-1, and GAT 3 (1:2000) from Abcam (Cambridge, UK); IL-1β (1:500) from R&D SYSTEMS (Minneapolis, USA); Arg-1 from BD Bioscience (NJ, USA); and glial fibrillary acidic protein (GFAP) (1:5000) from Sigma (St. Louis, MO, USA). As a control for protein loading, the same membranes were also incubated with anti-actin (Abcam, Cambridge, MA; 1:1000). Secondary antibodies were anti-rabbit, anti-goat, or anti-mouse IgG (1:2000) conjugated with alkaline phosphatase (Sigma, St. Louis, MO). The images were captured using the ScanJet 5300C (Hewlett- Packard, Amsterdam, the Netherlands) and band intensities quantified using the Alpha Imager 2200, version 3.1.2 (AlphaInnotech Corporation, San Francisco).
8
Western Blot Analysis of Neuroinflammatory Markers
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Tissue homogenates from the hippocampus and PFC were subjected to Western Blot (WB) analysis for the determination of protein levels. The extraction of protein and WB was performed as previously described [9 (link)–12 (link)]. The primary antibodies IL1b, IL 4, IL-18, TLR4, HMGB 1 and GAPDH were commercially obtained from Abcam, Cambridge, MA. Secondary antibodies were obtained: anti-mouse, 1:5000 dilution and anti-rabbit, 1:5000 dilution (SC-2314 and SC-2004 respectively, Santa Cruz Biotechnology, Santa Cruz, CA). The Immunoreactive bands were visualized using enhanced chemiluminescence (ECL Plus, Amersham), band intensities were quantified using ImageJ imaging software (NIH), and were normalized with GAPDH.
9
BALF Cytokine and Inflammatory Analysis
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One day after the end of sensitization, animals were sacrificed by i.p. administration of 50 mg/kg ketamine (CAS Number: 1867-66-9, Sigma Chemical Ltd., UK) and 5 mg/kg xylazine (CAS Number: 23076–35-9, Sigma Chemical Ltd., UK). The left lung was washed with one mL saline five times (5 mL totally). BALF was centrifuged at 2500 g at 4 °C for 10 min and supernatant was stored at − 70 °C [28 (link)] until analysis.
Cytokine and inflammatory levels including interleukin 4 (IL-4, Cat Number: ab100770, Abcam Company, Cambridge, MA, USA), interferon gamma (IFN-γ, Cat Number: ab46107, Abcam Company, Cambridge, MA, USA), immunoglobulin E (IgE, Cat Number: ab157736, Abcam Company, Cambridge, MA, USA), phospholipase A2 (PLA2, Cat Number: MBS262388, MyBioSource Inc., San Diego, California, USA) and total protein (TP, Cat Number: 128500, Pars Azmoon. Co., Iran) in the BALF were measured by enzyme-linked immunosorbent assay (ELISA) sandwich method with appropriate protocol recommended by company. The ratio of IFN-γ/IL4 as an index of Th1/Th2 was also calculated.
Cytokine and inflammatory levels including interleukin 4 (IL-4, Cat Number: ab100770, Abcam Company, Cambridge, MA, USA), interferon gamma (IFN-γ, Cat Number: ab46107, Abcam Company, Cambridge, MA, USA), immunoglobulin E (IgE, Cat Number: ab157736, Abcam Company, Cambridge, MA, USA), phospholipase A2 (PLA2, Cat Number: MBS262388, MyBioSource Inc., San Diego, California, USA) and total protein (TP, Cat Number: 128500, Pars Azmoon. Co., Iran) in the BALF were measured by enzyme-linked immunosorbent assay (ELISA) sandwich method with appropriate protocol recommended by company. The ratio of IFN-γ/IL4 as an index of Th1/Th2 was also calculated.
10
Microglia Polarization and Co-culture with Glioma Cells
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Interferon (IFN)-γ/lipopolysaccharide (LPS) and Interleukin (IL)-4 were applied to generate the M1 and M2 phenotypes of BV2 cell lines in vitro, respectively. LPS and IL-4 were from Abcam (Cambridge, MA, USA). BV2 cells (1 × 106 cells/mL) were seeded into the upper insert of a six-well Transwell plate (Corning Inc., Corning, MA, USA) and cultured at 37°C for 6 h, followed by incubation with either LPS (20 ng/mL) or IL-4 (20 ng/mL) for another 24 h at 37°C. LPS-polarized M1 and IL4-polarized M2 microglia were then washed with phosphate-buffered saline (PBS) and co-cultured with GL261 cells (2 × 105 cells per well) without direct contact for 48 h at 37°C. The co–cultured GL261 cells were then washed and harvested for subsequent experiments.
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