Western blotting was performed from neonatal rat cardiomyocytes using standard procedures. Antibodies used were METTL3 (Bethyl Laboratories), MAP3K6 (Novus Biologicals), MAP4K5 (Thermo Scientific Pierce), MAPK14 (Cell Signaling Technology) and GAPDH (Fitzgerald Industries). Masson’s trichrome staining was performed from histological sections generated from paraffin-embedded hearts. Immunostaining was performed using α-Actinin antibodies (Sigma Aldrich) and cell area was quantified using CellProfiler following published methods.24 (link) Detection of the cell membrane was performed using FITC-conjugated Wheat Germ Agglutinin (Sigma Aldrich). Cross-sectional area was measured using ImageJ. RNA was extracted from neonatal rat cardiomyocytes or mouse hearts using Trizol and reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Selected genes and m6A peaks were analyzed by real-time PCR using SYBR green (Applied Biosystems). Quantified mRNA expression was normalized to Rpl7 (Ribosomal Protein L7), and expressed relative to controls.
Fitc conjugated wheat germ agglutinin
Manufactured by Merck Group
Sourced in United States
FITC-conjugated wheat germ agglutinin is a fluorescently labeled lectin that binds to N-acetylglucosamine and sialic acid residues on cell surfaces. It is commonly used as a staining reagent in microscopy and flow cytometry applications to label and visualize cell surface glycoproteins.
Automatically generated - may contain errors
Lab products found in correlation
Sp2 confocal microscope, by Leica (2 mentions)
Apex antibody labelling kit, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Mettl3, by Fortis Life Sciences (1 mentions)
Map3k6, by Novus Biologicals (1 mentions)
Mapk14, by Cell Signaling Technology (1 mentions)
α actinin antibody, by Merck Group (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Map4k5, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Biosynth (1 mentions)
Confocal microscope, by Leica (1 mentions)
Eclipse ci s, by Nikon (1 mentions)
Dab kit, by ZSGB-BIO (1 mentions)
Masson trichrome, by Merck Group (1 mentions)
Dako antibody diluent, by Agilent Technologies (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
Dm2000, by Leica (1 mentions)
18 protocols using fitc conjugated wheat germ agglutinin
1
Western Blotting and Immunostaining of Neonatal Rat Cardiomyocytes
2
Cardiomyocyte Size Evaluation via WGA
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Cardiomyocyte size was evaluated using wheat germ agglutinin staining. The rat heart was fixed in 4% paraformaldehyde, and then, the frozen tissues were sectioned into 20 μm slides, rinsed with PBS and stained for cardiomyocyte membrane with FITC‐conjugated wheat germ agglutinin (Sigma). Finally, the heart cross section was imaged with Leica confocal microscope.
3
Immunohistochemical Analysis of Cardiac Proteins
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Sections for hematoxylin and eosin (H&E) and Masson trichrome staining were generated from paraffin-embedded hearts. Frozen sections were used to visualize cardiomyocyte cell membranes by staining with FITC-conjugated wheat germ agglutinin (Sigma-Aldrich, United States), as described before. For immunohistochemical staining, sections were deparaffinized in xylene and rehydrated. Antigen retrieval was performed with protease K at 37°C for 15 min. A solution of 3% H2O2 was used to block the activity of endogenous peroxidase. The sections were then incubated overnight at 4°C with WWP1 (1/100, Abcam, #ab43791) or DVL2 antibody (1/100, Cell Signaling Technology, #3224S). After three washes in PBS, biotinylated secondary antibodies were then added and incubated for 1 h at room temperature, followed by color development with DAB kit (ZSGB-Bio). Negative control experiments were done by omitting the primary antibodies. The sections were examined using a microscope (ECLIPSE Ci-S, Nikon).
4
Histological Analysis of Cardiac Morphology
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Histological analysis of the hearts was carried out as we have described previously.33 (link) Briefly, hearts were excised, fixed in 10% formalin, embedded in paraffin and sectioned into 7 μm slices, and stained with hematoxylin–eosin (HE). To measure the cross-sectional area of the cardiomyocytes, the sections were stained with FITC-conjugated wheat germ agglutinin (Sigma) according to the method previously described.37 (link) The myocardial fibrosis was determined by standard Masson trichrome staining in the heart sections according to the manufacturer's instructions (Sigma).
5
Visualizing Candida albicans Cell Wall
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C. albicans yeast cells were fixed directly in suspension for 1 h at 30°C by adding 37% formaldehyde solution to a final concentration of 3.7%. After fixation, cells were washed in phosphate-buffered saline (PBS; pH 7.4) and stained with 250 μg ml-1 FITC-conjugated wheat germ agglutinin (Sigma-Aldrich) for 30 min at room temperature. After washing of unbound FITC-WGA with PBS (pH 7.4), cells were immobilized on poly-L-lysine (0.1 mg ml-1, Sigma-Alrich) coated glass slides for 20 min at room temperature. Blocking was carried out in PBS (pH 7.4) with 2% bovine serum albumin for 1 h at room temperature or overnight at 4°C. For immunofluorescence, cells were stained with mouse monoclonal anti-V5 antibody (1:100; Clone SV5-PK1, Acris) in Dako antibody diluent (Agilent) for 1 h at room temperature and goat anti-mouse Alexa Fluor 555 antibody (1:400; Thermo Fisher) for 30 min at room temperature. Mowiol mounted slides were imaged using the 63x objective of a Zeiss Axio Observer Z1 microscope and Axiovision software.
6
Cardiomyocyte Cross-Sectional Analysis
7
Visualizing Actin and Cell Membrane Dynamics
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Transwell porous supports were excised and cell monolayers fixed (2.5% paraformaldehyde/PBS solution) prior to permeabilising (1% Triton X-100 in PBS) for staining filamentous actin (TRITC-phalloidin; Sigma-Aldrich) and DNA (DAPI; 4′,6-diamidino-2-phenylindole) as described7 (link). In some experiments FITC-conjugated wheat germ agglutinin (Sigma-Aldrich) was used to label the enterocyte surface membranes, following manufacturer’s instructions, while 1 μm latex beads (polystyrene, fluorescent yellow-green; Sigma-Aldrich) were also used as uptake substrates. Cells were examined by confocal microscope using a Leica SP-2 confocal microscope as described8 (link)9 (link).
8
Cardiomyocyte Cross-Sectional Area Quantification
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The cross-sectional area of individual cardiomyocytes was determined by staining 5 μm tissue sections with FITC-conjugated wheat germ agglutinin (catalog no.: L4895; Sigma–Aldrich) for 1 h at room temperature. The nuclei were counterstained with nuclear stain 4′,6-diamidino-2-phenylindole (catalog no.: D9542; Sigma–Aldrich). The images were taken by Leica DM2000 across multiple fields. Cell size was quantified using ImageJ (National Institutes of Health) software.
9
Histological Analysis of Cardiac Tissue
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Histological analysis was carried out as we previously described [40 (link)]. Briefly, hearts were excised, fixed in 10% formalin, embedded in paraffin and sectioned into 4-μm slices. The slides were stained with hematoxylin-eosin (H&E) and examined under a light microscope. The cross-sectional area of cardiomyocytes was determined by staining with FITC-conjugated wheat germ agglutinin (Sigma). Cardiac fibrosis was assessed by standard Masson trichrome staining (Sigma) following manufacturer’s instructions.
10
Cardiac Myocyte Morphometry via Fluorescence Imaging
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Heart tissue was fixed in 4% formaldehyde overnight, embedded in paraffin, serially sectioned at 10 µm intervals and then mounted on slides. Sections were stained following deparaffinization with FITC-conjugated wheat germ agglutinin (Sigma Aldrich Cat #L4895) and diluted to a 1:30 with phosphate-buffered saline (PBS). The sections were washed three times with PBS and mounted in Fluorescence Mounting Medium (Dako, S3023, Santa Clara, CA, USA). Images were acquired using 3DHistech Panoramic 250 Flash III (3DHISTECH Ltd., Budapest, Hungary). Cell size was analyzed by calculating the cross sectional area using Image J software.
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