Cells were lysed with RIPA lysis buffer and incubated on ice for 30 min. After centrifugation at 15 000 rpm for 5 min, supernatants were collected and mixed with 6× SDS sample buffer. The samples were boiled at 95 °C for 5 min and subjected to immunoblotting following standard procedures. The following primary antibodies were used for immunoblotting: mouse monoclonal Akt (40D4, Cell Signaling Technology; 1:1000), ERK1/2 (E10, Cell Signaling Technology; 1:1000), IL13Ralpha1 (D‐2, Santa Cruz Biotechnology; 1:1000), JNK (clone 37, BD Transduction Laboratories; 1:1000); rabbit monoclonal phospho‐Akt (Thr308) (C31E5E, Cell Signaling Technology; 1:1000), phospho‐Akt (D9E, Cell Signaling Technology; 1:1000), phospho‐ERK1/2 (D13.14.4E, Cell Signaling Technology; 1:1000), GAPDH‐HRP (14C10, Cell Signaling Technology; 1:5000), Gli1 (NB600‐600, Novus Biologicals; 1:1000), phospho‐JNK (81E11, Cell Signaling Technology; 1:1000), STAT6 (D3H4, Cell Signaling Technology; 1:1000), phospho‐STAT6 (D8S9Y, Cell Signaling Technology; 1:1000); rabbit polyclonal IFT88 (13967‐1‐AP, Proteintech; 1:1000), IL4R (bs‐2458R, Bioss; 1:1000), ST2 (ab25877, Abcam; 1:1000), and Trichoplein (home‐made; 1:1000). After staining with HRP‐conjugated secondary antibody (Molecular Probes), bands were detected using ECL chemiluminescence.
Ab25877
Manufactured by Abcam
Sourced in United States
Ab25877 is a primary antibody that can be used for immunohistochemistry (IHC) applications. It targets a specific protein. The product is suitable for use in human samples.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
Af114, by R&D Systems (2 mentions)
Mca497ga, by Abcam (2 mentions)
T7451, by R&D Systems (2 mentions)
Prb 160p, by Bio-Rad (2 mentions)
Eclipse nie, by Leica (2 mentions)
Af3626, by Abcam (2 mentions)
Af3626, by R&D Systems (2 mentions)
Epr2673, by Abcam (2 mentions)
Ab15580, by Abcam (2 mentions)
Pe or fitc conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Bx51 fluorescent microscope, by Olympus (2 mentions)
Ab118503, by Abcam (2 mentions)
Ab109223, by Abcam (2 mentions)
A32754, by Thermo Fisher Scientific (2 mentions)
A32723, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab45939, by Abcam (1 mentions)
15 protocols using ab25877
1
Immunoblotting Analysis of Cellular Signaling
2
Immunohistochemical Analysis of ST2 in Colonic Tissue
3
Immunohistochemical Analysis of ST2 in Colonic Tissue
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The colonic tissues were harvested, fixed with 4% paraformaldehyde and embedded in paraffin. The tissue samples were then cut into 4 µm thick sections. Rehydrated paraffin sections were boiled in a microwave oven for epitope retrieval using a sodium citrate buffer (pH 6, 10 min). Slides were stained with an anti-mouse ST2 antibody (Abcam, ab25877, 1:500 dilution) overnight at 4°C and then incubated with a rabbit HRP polyclonal antibody for 1 h at room temperature. HRP was then visualized with DAB (VECTOR).
4
Immunofluorescence Staining of Enteroids and Myofibroblasts
5
Multiparametric Immunostaining Protocol
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Paraffin slides were rehydrated by consecutive, descending
processing through Xylene and Ethanol (100%–50%). Paraffin slides
underwent citrate-based retrieval (Vector Unmasking), for frozen sections
only select panels were citrate retrieved.
Tissue sections were blocked and permeabilized for 30 – 60
minutes in 10% NDS, 4% BSA and 0.5% TritonX. Primary antibodies (Krt5 (rb)
Covance (Prb-160P) 1:1000; F4/80 (rt) Biorad (MCA497GA) 1:250; RFP (gt)
Abcam (ab25877) 1:500; acet.Tub (ms) Sigma (T7451) 1:2000; CD45 (gt) R&D
(AF114) 1:250) were diluted in primary block solution (2.5% NDS, 1% BSA,
0.125% TritonX) and incubated overnight at 4°C in a humidified
chamber. All fluorophore-conjugated secondary antibodies were diluted in
secondary block solution (5% NDS, 2% BSA, 0.25% TX) at 1:500 containing
Höechst nuclear stain dye. EdU staining was performed according to
manufacturer recommendations (Invitrogen). Images of sections were captured
on a Nikon Eclipse NiE or a Leica SP6.
processing through Xylene and Ethanol (100%–50%). Paraffin slides
underwent citrate-based retrieval (Vector Unmasking), for frozen sections
only select panels were citrate retrieved.
Tissue sections were blocked and permeabilized for 30 – 60
minutes in 10% NDS, 4% BSA and 0.5% TritonX. Primary antibodies (Krt5 (rb)
Covance (Prb-160P) 1:1000; F4/80 (rt) Biorad (MCA497GA) 1:250; RFP (gt)
Abcam (ab25877) 1:500; acet.Tub (ms) Sigma (T7451) 1:2000; CD45 (gt) R&D
(AF114) 1:250) were diluted in primary block solution (2.5% NDS, 1% BSA,
0.125% TritonX) and incubated overnight at 4°C in a humidified
chamber. All fluorophore-conjugated secondary antibodies were diluted in
secondary block solution (5% NDS, 2% BSA, 0.25% TX) at 1:500 containing
Höechst nuclear stain dye. EdU staining was performed according to
manufacturer recommendations (Invitrogen). Images of sections were captured
on a Nikon Eclipse NiE or a Leica SP6.
6
Immunofluorescent Labeling of IL-33+ Cells
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Standard immunofluorescent methods were applied for IL-33 IR cell types in the brains of Sham + PBS and RNS groups, which was described previously (Gao et al., 2017b (link)). Brain tissue was fixed by cardiac perfusion, and the brain was first perfused with PBS, then perfused with 4% paraformaldehyde, and cut into 5 μm sections using a cryostat. Briefly, (1) the slices were fixed in 4% paraformaldehyde for 10 min; (2) Incubated with 3% H2O2 for 5–10 min at room temperature to eliminate endogenous peroxidase activity; (3) Washed with PBS three times for 5 min each time, and placed the slices in a boiled sodium citrate solution for microwave repair for 20 min; (4) 0.5% Triton punched for about 10min; (5) Washed PBS three times for 5 min each time, 5% BSA was blocked for 2 h; (6) Incubated sections with anti-IL-33 (1:100; R&D, AF3626), anti-ST2 (1:200; Abcam, ab25877), anti-NeuN (1:200; Abcam), anti-Olig-2 (1:500; Millipore) diluted in blocking buffer, then the sections were incubated for 2 h at 4°C with an appropriate fluorescence-conjugated secondary antibody (1:200, Jackson Immuno-Research). Sections were stained for DAPI (1:5000, Beyotime Institute of Biotechnology) to visualize the nucleus. Images were captured with a fluorescence microscope (Zeiss).
7
Multiparametric Immunostaining Protocol
8
Immunohistochemical Analysis of Mouse Brain
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Mouse pups were anesthetized and transcardially perfused with 4% paraformaldehyde in PBS. The brain tissues were embedded in paraffin and sliced coronally in 5 μm. Tissue sections were dewaxed, quenched with 3% hydrogen peroxide for 10 min, and incubated with 5% bovine serum albumin (BSA) for 30 min. The sections were stained overnight at 4 °C with either anti-ST2 (1:200, ab25877, Abcam), anti-IL-33 (1:500, AF3626, R&D), anti-GFAP (1:500, 104805-T08, Sino Biological Inc.), anti-Olig2 (1:500, EPR2673, Abcam), anti-Iba1(1:50, 20A12.1, Sigma-Aldrich), anti-Neun (1:50, A60, Sigma-Aldrich), or anti-Ki67 (1:500, ab15580, Abcam) followed by PE- or FITC-conjugated secondary antibodies (eBioscience). TUNEL staining was performed with the in situ cell death detection kit (Roche). The slides were counterstained with nuclear dye DAPI and observed on an Olympus BX51 fluorescent microscope.
9
IL-33 Signaling Pathway Inhibition
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Recombinant human IL-33 (3625-IL) and anti-IL-33 antibody (AF3625) were purchased from R&D Systems (Minneapolis, MN, USA). The anti-TNC antibody (ab108930) and anti-ST2 antibody (ab25877) were purchased from Abcam (Cambridge, MA, USA). The specific inhibitors of PI3K/AKT (LY294002 #9901) and MAPK/ERK (PD98059 #9900) were acquired from Cell Signaling Technology, Inc. (Danvers, MA, USA), and the inhibitor of NF-κB (BAY11-7082 B5556) and antibody for β-actin were purchased from Sigma-Aldrich (St Louis, MO, USA). The phospho-specific and total antibodies against PI3K, ERK1/2 and NF-κB were also purchased from Sigma-Aldrich.
10
Protein Expression Analysis of IL-33, sST2, and TCF7L2
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Total proteins were extracted from mouse heart tissues and H9C2 cells using RIPA lysis buffer. Proteins from the serum of participants were extracted using the serum protein extraction kit (Solarbio, Beijing, China). After concentration was measured using a BCA protein assay kit (Solarbio), the protein samples were run on 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking, the membranes were incubated with primary antibodies targeting IL-33 (ab118503; Abcam, Cambridge, UK), sST2 (ab25877; Abcam), TCF7L2 (sc-271287, Santa Cruz Biotechnology, Santa Cruz, CA, USA), GAPDH (ab9485; Abcam) at 4°C overnight, followed by incubation with anti-rabbit (ab6721; Abcam) or anti-mouse (ab205719; Abcam) conjugated to horse radish peroxidase at 25°C for 1 h. The band signals were visualized using an ECL substrate (Solarbio).
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