Preparation of cryostat or paraffin tissue sections was done as described previously.39 (link) After routine processing, liver sections 7 μm thick were stained with H&E for histological analysis or saturated picric acid containing 0.1% Sirius Red and 0.1% Fast Green for collagen deposition. Paraffin sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), and frozen liver sections were incubated with anti-CD8+, anti-CD4+, anti-F4/80, anti-Gr-1 antibodies (BD PharMingen, San Diego, CA), respectively, and then were incubated with species matched Alexa dye-labeled or horseradish peroxidase (HRP) conjugated secondary antibodies. Frozen liver sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), anti-CD11b (BD PharMingen, San Diego, CA), anti-F4/80 (BD PharMingen, San Diego, CA), anti-α-SMA (Abcam, Cambridge, UK) antibodies, followed by the staining with Alexa Fluor 488 or 555-conjugated secondary antibodies (Invitrogen, Grand Island, NY). Sections were evaluated under the microscope (DP71, OLYMPUS) for bright-field and fluorescence microscopy.
Anti s100a4
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-S100A4 is a lab equipment product that recognizes the S100A4 protein, also known as metastasin or fibroblast-specific protein 1 (FSP1). S100A4 is a member of the S100 family of calcium-binding proteins and is involved in a variety of cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti α sma, by Abcam (6 mentions)
Alexa fluor 488 or 555 conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
Anti f4 80, by BD (3 mentions)
Anti cd11b, by BD (2 mentions)
Anti cx43, by BD (2 mentions)
Elastase, by Roche (2 mentions)
Anti nk1, by BioLegend (2 mentions)
Anti f4 80, by BioLegend (2 mentions)
Anti cd31, by BioLegend (2 mentions)
Anti cd3, by BioLegend (2 mentions)
Anti sp c, by ABclonal (2 mentions)
Anti cd45, by BioLegend (2 mentions)
Anti epcam, by BioLegend (2 mentions)
Anti ly6g, by BioLegend (2 mentions)
Anti β actin, by Abcam (2 mentions)
Gapdh, by Fujifilm (2 mentions)
Mouse monoclonal anti ki 67, by Agilent Technologies (1 mentions)
Anti cx43, by Abcam (1 mentions)
Anti cx40, by Abcam (1 mentions)
Anti α smooth muscle actin, by Abcam (1 mentions)
23 protocols using anti s100a4
1
Histological Analysis of Liver Sections
2
Tumor Immunohistochemistry and Microscopy
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A section of each tumor was fixed in 4% paraformaldehyde and then embedded in paraffin for histological hematoxylin–eosin (H&E) (Zhongshanjinqiao, Beijing, China), anti-S100A4 (Abcam, Cambridge, UK) and anti-Ki67 (BD Pharmingen, San Diego, CA) immunohistochemical staining.
For double staining, frozen tumor sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), anti-CD11b (BD Pharmingen, San Diego, CA), anti-F4/80 (BD Pharmingen, San Diego, CA) and anti-α-SMA (Abcam, Cambridge, UK) antibodies, followed by staining with Alexa Fluor 488- or 555-conjugated secondary antibodies (Invitrogen, Grand Island, NY). Sections were evaluated under the microscope (DP71, OLYMPUS) using both bright-field and fluorescence microscopy.
For double staining, frozen tumor sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), anti-CD11b (BD Pharmingen, San Diego, CA), anti-F4/80 (BD Pharmingen, San Diego, CA) and anti-α-SMA (Abcam, Cambridge, UK) antibodies, followed by staining with Alexa Fluor 488- or 555-conjugated secondary antibodies (Invitrogen, Grand Island, NY). Sections were evaluated under the microscope (DP71, OLYMPUS) using both bright-field and fluorescence microscopy.
3
Histological Analysis of Adipose and Liver Tissues
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Cryostat sections from white adipose tissues (WATs) and liver tissues were prepared. Five micrometer-thick sections were stained with H&E for histological analysis or Oil Red O for the fat droplets accumulation.
For immunohistochemistry (IHC) staining, cryostat sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), anti-F4/80 (BD Pharmingen, San Diego, CA), and anti-Gr-1 antibodies (BD Pharmingen, San Diego, CA), respectively, then were incubated with species matched Alexa dye-labeled or horseradish peroxidase-conjugated secondary antibodies.
For fluorescence double staining, cryostat sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), anti-F4/80 (BD Pharmingen, San Diego, CA), anti-α-SMA (Abcam, Cambridge, UK) antibodies, followed by the staining with Alexa Fluor 488 or 555-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Sections were evaluated under the microscope (DP71, OLYMPUS) for bright-field and fluorescence microscopy.
For immunohistochemistry (IHC) staining, cryostat sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), anti-F4/80 (BD Pharmingen, San Diego, CA), and anti-Gr-1 antibodies (BD Pharmingen, San Diego, CA), respectively, then were incubated with species matched Alexa dye-labeled or horseradish peroxidase-conjugated secondary antibodies.
For fluorescence double staining, cryostat sections were incubated with anti-S100A4 (Abcam, Cambridge, UK), anti-F4/80 (BD Pharmingen, San Diego, CA), anti-α-SMA (Abcam, Cambridge, UK) antibodies, followed by the staining with Alexa Fluor 488 or 555-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Sections were evaluated under the microscope (DP71, OLYMPUS) for bright-field and fluorescence microscopy.
4
Prostate Cancer Immunohistochemistry Protocol
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Immunohistochemistry of human prostate cancer tissues and mouse tumor tissue were carried out as described previously [16 (link)] with rabbit monoclonal anti-S100A4 (Abcam, Cambridge, UK) or mouse monoclonal anti-Ki67 (DAKO, Santa Clara, CA, USA) used as primary antibodies respectively. The study using the patient-derived tissues was approved by the research ethics committees of Niigata University Medical and Dental Hospital. Informed consent was obtained from each patient for use of the materials.
5
Immunohistochemical Analysis of Porcine SMCs
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Cultured porcine SMCs were washed twice with phosphate-buffered saline (PBS; Corning, New York, NY, USA) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. Subsequent to incubation with primary (anti-Cx40, anti-Cx43, anti-S100A4 and anti-α-smooth muscle actin (SMA); Abcam, Cambridge, MA, USA) and polymer helper and poly-peroxidase anti-mouse/rabbit immunoglobulin G secondary antibodies (PV-9000 kit; GBI Labs, Mukilteo, WA USA) were added for 1 h, and diaminobenzidine (Roche Diagnostics GmbH) was used for signal detection. The slides were washed under running water and tissue samples were counterstained with hematoxylin. Sections were observed and imaged using an Olympus CX31 microscope (Olympus Corporation, Tokyo, Japan).
6
Quantifying S100A4 and Sphingolipids in BALF and Serum
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S100A4 protein levels in human BALF were detected by ELISA. In brief, 96-well plates (Corning, Tewksbury, MA, USA) were coated with anti-S100A4 (1 µg/ml; clone 3B11) (Purified by Cwbio, Beijing, China) in carbonate solution (pH 9.6). BALF samples, diluted 1:5 with phosphate-buffered saline (pH 7.4) containing 3% bovine serum albumin (Thermo Fisher Scientific, Waltham, MA, USA) were incubated for 1 h at 37°C. Captured antigen was detected with rabbit polyclonal anti-S100A4 (1:1,000; Abcam, Cambridge, UK). The complex was visualized by horseradish peroxidase-conjugated anti-rabbit IgG (1:3,000; Sigma-Aldrich, St. Louis, MO, USA) and TMB substrate (Cwbio). Absorbance was detected using a microplate reader (EPOCH12; Instruments, Winooski, VT, USA). Serum concentrations were calculated from a standard curve of recombinant human S100A4 (R&D Systems, Minneapolis, MN, USA) in a concentration range of 12.5–1,000 ng/ml.
Blood was collected from eyeball venous plexus of mice, and the serum stored at 4°C. S1P or SPH was detected by ELISA kits (Enzyme-Linked Biotechnology, Shanghai, China) according to the manufacturer’s instructions.
Blood was collected from eyeball venous plexus of mice, and the serum stored at 4°C. S1P or SPH was detected by ELISA kits (Enzyme-Linked Biotechnology, Shanghai, China) according to the manufacturer’s instructions.
7
Immunohistochemical Analysis of Angptl2 and TGF-β1 in Tissues
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LF tissue samples were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned. After the sections were pre-treated with Target Retrieval Solution, pH 9 (Tris/EDTA buffer, pH 9; Dako Japan, Co., Ltd., Tokyo, Japan), endogenous peroxidases were blocked using periodic acid (Nichirei, Tokyo, Japan). We used the following antibodies as primary antibodies: anti-human Angptl2 antibody [19] (link), anti-vimentin, anti-CD3, anti-CD15, anti-CD20, anti-CD68 (Dako Japan), anti-S100A4 (Abcam, Cambridge, UK), and anti-human p-Smad3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After treatment using EnVision + System-HRP-labeled Polymer (Dako Japan), the labeling was visualized using a Histofine 3,3′-diaminobenzidine (DAB) kit (Nichirei). For double immunofluorescent staining, anti-vimentin (Dako Japan) and anti-Angptl2 or anti-TGF-β1 (Abcam) were used as the primary antibodies, and Alexa Fluor 488-labeled anti-rabbit IgG and Alexa Fluor 594-labeled anti-mouse IgG (Life Technologies) were used as the secondary antibodies. Nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole (DAPI).
8
Tissue Microarray-Based Evaluation of S100A4 in HCC
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Tissue microarrays (TMAs) consisting 75 HCC patient cases for immunohistochemistry were purchased from Xin Chao (Shanghai, China). The company provided ethical statement to confirm that the local ethics committees approved their consent procedures and all study participants signed an approved informed consent form. The ethical statement provided by the company and the protocol of experiment had been checked carefully and approved by Ethics Committee of Beijing Jiaotong University. The tissue microarrays were stained with anti-S100A4 (Abcam, Cambridge, UK). In the 75 cases, was carried out for evaluating S100A4 staining according to previous study.48 The intensity of S100A4 expression was scored as follows: 0, negative; 1, weak; 2, moderate; 3, strong (Figure 1(a )). Extent of staining was scored as follows: 1, 0 to 25%; 2, 25 to 50%; 3, 50 to 75% or 4, 75 to 100%. Five random fields were observed under a light microscope. The final score was determined by multiplying the scores of intensity with the extent of staining. The sum from 0 to 5 was defined as S100A4 negative. The sum from 5 to 45 was defined as S100A4 low expression. The sum from 45 to 80 was defined as S100A4 high expression.
9
S100A4 Protein Interaction Assay
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Endothelial cells were seeded in 10-cm dishes, followed by stimulation (37°C) with or without 100 µM H2O2 for 48 h. Subsequently, the HepG2 cell (Tianjin Saierbio Biotechnology Co., Ltd., Tianjin, China) lysates were prepared with 1% Tris-Triton cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) pre-mixed with 1 mM phenylmethanesulfonyl fluoride on ice for 30 min and centrifuged (4°C) at 12,000 × g for 30 min. The supernatants were incubated (4°C) overnight with 30 µl Dynabeads protein A or protein G (Thermo Fisher Scientific, Inc.) pre-coated with anti-S100A4 (1:250, Abcam) antibodies. The immunocomplexes were subjected to western blot analysis. The normal corresponding immunoglobulin G control was assayed simultaneously.
10
Protein Expression in MB49 and MCSCs
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The MB49 cells and MCSCs were respectively harvested. Equal amount proteins were extracted from cells, and separated by 10% sodium dodecyl sulfate -polyacrylamide gel electrophoresis followed by transferring to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked with 5% skim milk in PBS, and incubated overnight at 4 °C with the primary antibodies including anti-S100A4 (Abcam, Cambridge, MA), anti- IKK (Abcam) and anti-β-actin antibody (Abcam) followed by secondary antibodies (Abcam). Bands were visualized using Fluor Chem FC2 (Alpha Innotech, San Leandro, CA).
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