Paraffin-embedded biopsy specimens and 3D organoid products were serially sectioned and subjected to H&E staining or IHC as described previously.12 (link),18 (link) In brief, sections were incubated overnight at 4°C with anti-CD73 antibody (rabbit monoclonal, ab133582, 1:100; Abcam, Cambridge, UK) and anti–Itg-β4 (mouse monoclonal, MAB4060, 1:100; R&D Systems, Minneapolis, MN) for immunofluorescence, and anti–Ki-67 (rabbit monoclonal, ab16667, 1:200; Abcam), anti-SOX2 (rabbit monoclonal, 14962S, 1:300; Cell Signaling Technology, Danvers, MA), or anti-STAT6 (phospho Y641, rabbit polyclonal, ab28829, 1:50; Abcam) for IHC. IHC of 3D organoids for CD73 and CD104 was performed by BOND RXm (Leica Biosystems, Nusslock, Germany) with anti-CD73 antibody (rabbit polyclonal, ab175396, 1:200; Abcam) or anti-CD104 monoclonal antibody (NBP2-37392, 1:1000, Novus Biologicals, Englewood, CA). Imaging was performed with the BZ-X710 Fluorescence Microscope (Keyence, Osaka, Japan).
Ab28829
Manufactured by Abcam
Sourced in United States
Ab28829 is a lab equipment product offered by Abcam. It serves as a tool for researchers, but a detailed description without interpretation or extrapolation is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab32520, by Abcam (6 mentions)
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Ab203883, by Thermo Fisher Scientific (3 mentions)
Anti ifnγ 500 p119, by Thermo Fisher Scientific (3 mentions)
Ab16030, by Abcam (3 mentions)
Ab3987, by Abcam (3 mentions)
Ab11524, by Abcam (3 mentions)
Ab30645, by Abcam (3 mentions)
Ab64693, by Abcam (3 mentions)
Ab15323, by Abcam (3 mentions)
Ab44718, by Abcam (2 mentions)
Sc 271430, by Santa Cruz Biotechnology (2 mentions)
Bz x710 fluorescence microscope, by Keyence (1 mentions)
Bond rxm, by Leica (1 mentions)
Ab133582, by Abcam (1 mentions)
Ab175396, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Luminate forte western hrp substrate, by Merck Group (1 mentions)
Anti rabbit igg na934, by GE Healthcare (1 mentions)
11 protocols using ab28829
1
Immunohistochemical Analysis of 3D Organoids
2
Western Blot Analysis of STAT6 Phosphorylation
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Total cell lysates were extracted as previously described using a lysis buffer (#9803; Cell Signaling Technology).27 Following electrophoresis, the membrane with separated proteins was blocked with PBS containing 5% skimmed milk powder for 2 hours at room temperature, and then incubated at 4°C overnight with anti‐human STAT6 rabbit polyclonal Ab (ab44718, 1:200; Abcam), anti‐human phospho‐STAT6 rabbit polyclonal Ab (ab28829, 1:100; Abcam), or anti‐human β‐actin mouse mAb ( #612656, 1:200; BD Biosciences, Tokyo, Japan). After overnight incubation, membranes were incubated for 30 minutes with an HRP‐conjugated anti‐mouse IgG (W4011, 1:2,500; Promega, Tokyo, Japan) or anti‑rabbit IgG (NA934, 1:300; GE Healthcare, Tokyo, Japan). Each complex was detected using a Luminate Forte Western HRP Substrate (Merck Millipore) according to the manufacturer's instructions.
3
Western Blot Analysis of THP-1 Cell Signaling
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THP-1 cells were rinsed twice with ice-cold PBS and lysed with RIPA buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris pH 7.4, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail). The protein concentration of the cell lysates was measured using the Bradford reagent (Thermo Fisher, USA). Next, 30 μg of each protein sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose (NC) paper. The resultant NC papers were then incubated overnight at 4 °C with a range of specific primary antibodies, including signal transducer and activator of transcription (STAT6) (1:1000, ab32520, Abcam, USA), p-STAT6 (1:1000, ab28829, Abcam, USA), peroxisome proliferator-activated receptor γ (PPARγ) (1:1000, #2435, Cell Signaling, USA), high mobility group protein B1 (HMGB1) (1:5000, ab18256, Abcam, USA), and interleukin-4 receptor (IL-4 R) (1:1000, ab203398, Abcam, USA). After incubation with the indicated primary antibody, the proteins of interest were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Millipore, USA) and visualized by the ECL detection system (Millipore, USA).
4
Protein Expression Profiling of Signaling Pathways
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Cells or tissues were harvested at indicated times and homogenized in ice-cold suspension buffer supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using the BCA Protein assay kit (Thermo Scientific, Waltham, MA). Equal amounts of protein were fractionated by SDS polyacrylamide gels, followed by immunoblotting with the following primary antibodies: NF-κB Pathway Sampler Kit (diluted at 1:1000, 9936, CST, MA), phospho-MAPK Family Antibody Sampler Kit (diluted at 1:1000, 9910, CST, MA), MAPK Family Antibody Sampler Kit (diluted at 1:1000, 9926, CST, MA), phospho-Stat6 antibody (Rabbit polyclonal, diluted at 1:1000, ab28829, Abcam), Stat6 antibody (Rabbit polyclonal, diluted at 1:1000, ab44718, Abcam), TRAF6 antibody (Rabbit polyclonal, diluted at 1:1000, ab94720, Abcam), TLR4 antibody (Rabbit polyclonal, diluted at 1:1000, ab13556, Abcam), CD14 antibody (Rabbit polyclonal, diluted at 1:1000, ab203294, Abcam), Myd88 antibody (Rabbit polyclonal, diluted at 1:1000, ab2064, Abcam), PPARγ antibody (Rabbit monoclonal (C26H12), diluted at 1:1000, sc-7273, Santa Cruz Biotechnology). Membranes were then incubated with peroxidase-conjugated secondary antibody, and specific bands were detected with a Bio-Rad (Hercules, CA) imaging system. Original blots were provided in Supplementary Fig. 8 .
5
Characterization of M1 and M2 Macrophages
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Macrophages were fixed with 4% paraformaldehyde and 1% glutaraldehyde for 10 min. Followed by rinsing and blocking with 2% BSA, macrophages were stained with primary antibodies for M1 markers CD68 (Abcam ab955) and CD86 (Abcam, ab53004) and M2 markers CD163 (Abcam, ab87099), CD206 (Abcam, ab8918), and phosphorylated STAT6 (Abcam, ab28829). Secondary antibody staining was done with Alexa Fluor 488, goat anti-mouse IgG (Abcam, ab150113) and Cy5, and goat anti-rabbit IgG (Abcam, ab6563). PROTOCOL Hema 3 staining systems (Fisher Scientific) were used to stain macrophage morphology according to the manufacturer’s protocol.
6
STAT Expression and Phosphorylation in Lung Tissue
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The expression levels of STAT1, STAT4, STAT6 and the corresponding phosphorylated proteins in lung digests were analyzed by Western blot analysis. Cell lysates (40 μg) were separated by 10% sodium dodecyl sulfate-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Roche, USA). After incubation in a blocking buffer containing 5% skim milk in TBST (12.5 mM Tris–HCl pH 7.5, 68.5 mM NaCl, and 0.1% Tween 20) for 1 h, the blots were incubated overnight with primary antibodies including rabbit anti-STAT1 (D1K9Y, Cell Signaling Technology, USA), rabbit anti-phosphorylated STAT1 (Tyr701, Cell Signaling Technology, USA), rabbit anti-STAT4 (C46B10, Cell Signaling Technology, USA), rabbit anti-phosphorylated STAT4 (ab28815, Abcam, UK), rabbit anti-STAT6 (ab32520, Abcam, UK), and rabbit anti-phosphorylated STAT6 (ab28829, Abcam, UK). An anti-mouse GAPDH antibody was used as a loading control. The blots were washed with TBST, incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit Ig (Jackson ImmunoResearch) and then developed with an enhanced chemiluminescence (ECL) substrate solution (Millipore).
7
Analyzing CLL-Infiltrated Lymph Nodes
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Lymph node biopsies of CLL‐infiltrated and tumour‐free samples were formalin‐fixed, paraffin‐embedded, arranged in Tissue Microarrays and stained for pSTAT6 (ab28829, Abcam) and pIRAK4 (ab216513, Abcam). The slides were analysed using Qupath (Bankhead et al, 2017 (link)) and the recommended protocol.
8
Immunoblotting Assay for Macrophage Markers
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Immunoblotting was performed as previously described [48 (link)] using the following primary antibodies: anti-iNOS (ab15323, Abcam), anti-CD16 (ab203883), anti-IFNγ (500-P119, PeproTech, Rocky Hill, NJ, USA), anti-Arg1 (sc-271430, Santa Cruz), anti-CD206 (ab64693, Abcam), anti-IL-4 (ab11524, Abcam), anti-STAT1 (ab3987, Abcam), anti-p-STAT1 (ab30645, Abcam), anti-STAT6 (ab32520, Abcam), anti-p-STAT6 (ab28829, Abcam), anti-SOCS3 (ab16030, Abcam), and the HRP-conjugated secondary antibody (1:5000). The plots were visualized by ECL Plus (Thermo).
9
Western Blot Analysis of STAT6 Activation
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When the cells were prepared, RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) was added to isolate the total protein. Fifty microgram of total protein was separated through SDS-PAGE at 100 V for 2 h in the 12% polyacrylamide gel. It was then electrically transferred to a polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% skim milk for 1 h at room temperature, the membrane was rinsed 3 times with TBST for 10 min each time. Antibodies specific to STAT6 (ab32520; Abcam), p-STAT6 (ab28829; Abcam) (concentration 1:1000) were added to incubate the membrane overnight at 4 °C. After the incubation, the membrane was rinsed with TBST; the membrane then was incubated with horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibody (concentration 1:3000) for 1 h at room temperature. Subsequently, the membrane was rinsed for 3 times with TBST for 10 min each time. Ultimately, the imaging was performed with the application of hypersensitive ECL (Amersham Pharmacia Biotech, Little Chalfont, UK), and the grayscale of each protein was analyzed by software Image J.
10
Immunoblotting for Macrophage Markers
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Immunoblotting was performed as previous describe [48] using the following primary antibodies: anti-iNOS (ab15323, Abcam,), anti-CD16 (ab203883)), anti-IFNγ (500-P119, PeproTech, Rocky Hill, NJ, USA), anti-Arg1 (sc-271430, santa cruz), anti-CD206 (ab64693, abcam), anti-IL-4 (ab11524, Abcam), anti-STAT1 (ab3987, Abcam), anti-p-STAT1 (ab30645, Abcam), anti-STAT6 (ab32520, Abcam), anti-p-STAT6 (ab28829, Abcam), anti-SOCS3 (ab16030, Abcam), and the HRP-conjugated secondary antibody (1:5000). The plots were visualized by ECL Plus (Thermo).
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