Postnatal day 15 wild-type and Alms1Gt/Gt mouse hearts were fixed with methanol, paraffin embedded, sectioned, deparaffinized, rehydrated, and subjected to citrate-based heat-mediated antigen retrieval. Slides were incubated with 5 μg/ml Alexa Fluor 647-conjugated wheat germ agglutinin (Invitrogen) overnight at 4 °C and mounted in Vectashield containing DAPI (Vector Labs, CA). Image acquisition was performed on an EVOS epifluorescence microscope (Life Technologies). Cardiomyocyte cross sectional area was measured using an automated algorithm with NIH Image J 1.47i software analyzing 300 – 600 cells from 3–4 areas per mouse heart.
Alexa fluor 647 conjugated wheat germ agglutinin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 647-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 cell surface marker in fluorescence microscopy and flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Evos epifluorescence microscope, by Thermo Fisher Scientific (2 mentions)
Vectashield containing dapi, by Vector Laboratories (2 mentions)
Lsm 510 meta laser scanning microscope, by Zeiss (2 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Metamorph acquisition software, by Molecular Devices (1 mentions)
Latrunculin a, by Merck Group (1 mentions)
Coolsnap hq2 ccd camera, by Teledyne (1 mentions)
Ti e microscope, by Yokogawa (1 mentions)
Prolong gold mounting media, by Thermo Fisher Scientific (1 mentions)
Ab76956, by Abcam (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
Ab18251, by Abcam (1 mentions)
Ar 1 laser scanning confocal microscope, by Nikon (1 mentions)
Taxol, by Merck Group (1 mentions)
Alexa fluor 488 igg, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Chamber slide, by Ibidi (1 mentions)
Hcx pl apo cs 63 1.40 0 60 oil objective, by Leica (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
10 protocols using alexa fluor 647 conjugated wheat germ agglutinin
1
Cardiomyocyte Morphometry in Mouse Hearts
2
Quantification of Cardiac Fibrosis and Myocyte Size
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Myocardium was fixed with 10% formaldehyde, paraffin embedded and sectioned into 4 μm slices. Masson’s trichrome staining was used to visualize collagen. Quantification of fibrosis content was performed in 4–6 regions of each heart. Wheat germ agglutinin staining of mouse heart sections: Slides were deparaffinized, rehydrated, and subjected to citrate-based heat-mediated antigen retrieval. Slides were incubated with 5 μg/ml Alexa Fluor 647-conjugated wheat germ agglutinin (Invitrogen) overnight at 4 °C and mounted using Prolong Gold mounting medium (Invitrogen)27 (link). Myocyte cross sectional area was measured using an automated algorithm with NIH Image J 1.47i software. Image acquisition was performed on a Zeiss LSM510-META laser scanning microscope.
3
Cardiomyocyte Morphometry in Mouse Hearts
4
Quantification of Cardiac Fibrosis and Myocyte Size
5
Visualizing Cytoskeletal Organization in NIH 3T3 Cells
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NIH 3T3 fibroblasts (ATCC) were cultured in DMEM media and supplemented with 10% FBS, 2 mM L-glutamine, and penicillin-streptomycin. Cells were plated on glass coverslips for 24 h before fixation. Cells treated with latrunculin A (Sigma-Aldrich) were incubated with the drug at 1 µM for 15 min before fixation. Cells were fixed with 4% paraformaldehyde for 15 min, washed 3× in PBS, and then incubated for 10 min with 5 µg/ml Alexa Fluor 647 conjugated wheat germ agglutinin (Invitrogen) to label the membrane. Cells were washed 3× in PBS, permeabilized with 0.5% Triton X-100, and then incubated with Alexa Fluor 488 conjugated phalloidin (Invitrogen) and a rabbit anti–human Septin 7 primary antibody (IBL-America), followed by a goat anti–rabbit Alexa Fluor 567 (Invitrogen) secondary antibody. Coverslips were mounted on glass slides in ProLong Gold mounting media (Invitrogen).
Cells were imaged on an inverted Nikon Ti-E microscope with a Yokogawa CSU-X spinning disk scanhead, a laser merge module containing 491, 561, and 642 laser lines (Spectral Applied Research), and an HQ2 CoolSNAP CCD camera (Roper Scientific). Metamorph acquisition software (Molecular Devices) was used to control the microscope hardware. Images were acquired with a Nikon 60× 1.49 NA ApoTIRF oil-immersion objective.
Cells were imaged on an inverted Nikon Ti-E microscope with a Yokogawa CSU-X spinning disk scanhead, a laser merge module containing 491, 561, and 642 laser lines (Spectral Applied Research), and an HQ2 CoolSNAP CCD camera (Roper Scientific). Metamorph acquisition software (Molecular Devices) was used to control the microscope hardware. Images were acquired with a Nikon 60× 1.49 NA ApoTIRF oil-immersion objective.
6
VSMC Calcification and Microtubule Dynamics
7
Immunofluorescence Imaging of Ovaries
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Five to ten ovary pairs were dissected into ice-cold PBS and fixed in IF Fixing Buffer (4% formaldehyde, 0.3% Triton X-100, 1x PBS) for 20 min at room temperature with rotation. Fixed ovaries were washed thrice with PBX (0.3% Triton X-100, 1x PBS), 10 min each wash, and incubated in BBX (0.1% BSA, 0.3% Triton X-100, 1x PBS) for 30 min for blocking. Primary antibodies diluted in BBX were added to ovaries and binding was performed at 4°C overnight. After three 10 min-washes in PBX, ovaries were incubated with secondary antibodies (1:1000 dilution in BBX) at 4°C overnight. Afterwards, the ovaries were washed 4 times with PBX, with the second wash done with DAPI (1:50,000 dilution). To visualize the nuclear envelope, Alexa Fluor 647-conjugated wheat germ agglutinin (1:200 dilution in PBX; Thermo Fisher Scientific) was added after DAPI staining for 20 min. Ovaries were finally mounted in ∼40 µl Prolong Diamond mounting medium and imaged on a Zeiss LSM-880 confocal-microscope with AiryScan detector. The resulting images were processed using FIJI/ImageJ (Schindelin et al. 2012 (link)). Supplementary Table S3 lists antibodies used in this study.
8
Visualizing Bacterial Engulfment by Neutrophils
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For the time-course experiments, Peri mCherry/ Cyto GFP E. coli were grown in the presence of L-arabinose as described above, washed three times in PBS and concentrated to OD 600 ~2 in PBS. Bacteria were immobilized on a poly-L-lysine (0.01%, Sigma-Aldrich) covered 8 well µ-slide chamber (Ibidi) for 45 minutes. Chambers were rinsed three times with PBS after which RPMI-HSA containing 1 µM To-pro-3 (Thermofisher) was added. A T=0 image was taken, after which 5% normal serum or Δlysozyme serum with or without 5 µg/ml purified lysozyme was added. The Δlysozyme serum + lysozyme was imaged with and without 20 µg/ml OmCI and Eculizumab. GFP and To-pro-3 intensity was measured after the indicated time-pointes at room temperature. To image GFP bacteria inside neutrophils, the samples were prepared as described above and fixed in 1.5% paraformaldehyde. Neutrophil membranes were stained for 15 minutes with 2 µg/ml Alexa Fluor 647-conjugated Wheat Germ Agglutinin (ThermoFisher). Samples were concentrated and dried onto 1% agar pads.
All images were obtained using a Leica SP5 confocal microscope with a HCX PL APO CS 63×/1.40-0.60 OIL objective (Leica Microsystems, the Netherlands). GFP was measured using the 488 laser, Alexa-647 and To-pro-3 were imaged using the 647 laser. Both lasers were used in combination with the appropriate emission filter settings.
All images were obtained using a Leica SP5 confocal microscope with a HCX PL APO CS 63×/1.40-0.60 OIL objective (Leica Microsystems, the Netherlands). GFP was measured using the 488 laser, Alexa-647 and To-pro-3 were imaged using the 647 laser. Both lasers were used in combination with the appropriate emission filter settings.
9
Comprehensive Cellular Reagent Protocol
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Nocodazole, Cytocholasine B, Ficoll 400, puromycin, geneticin (G418), Hoechst 33342, dimethyl pimelimidate dihydrochloride, and all-trans-retinoic acid were from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 3000 was from Invitrogen (Carlsbad, CA, USA). Protein A Sepharose CL-4B and Glutathione Sepharose 4 Fast Flow were from GE Healthcare Life Sciences (Chicago, IL, USA). Protease-inhibitor mixture tablets (Complete EDTA-free) were from Roche Molecular Biochemicals (Mannheim, Germany). Restriction enzymes were from New England Biolabs (Ipswich, MA, USA). Oligonucleotides were synthesized by Sigma-Aldrich. Oligopeptides EYHAATRPDYISWGTQ (human γ-tubulin amino acid [aa] sequence 434–449) and EEFATEGTDRKDVFFY (human γ-tubulin aa sequence 38–53) [28 (link)] were synthesized by Jerini Peptide Technologies (Berlin, Germany). ER-Tracker Green (BODIPY FL Glibenclamide), AlexaFluor 555-conjugated, and AlexaFluor 647-conjugated wheat germ agglutinin (WGA) were from Thermo Fisher Scientific (Waltham, MA, USA). Purified C-terminally FLAG-tagged C53 and nucleophosmin were from OriGene Technologies (Rockville, MD, USA). Tunicamycin was from Sigma-Aldrich, and 1 mg/mL stock was prepared in DMSO.
10
Fluorescent Labeling of Cellular Organelles
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HeLa cells were obtained from the American Type Culture Collection and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Nacalai Tesque, #08458-45) containing 10% fetal bovine serum (Equitech Bio, #268-1). Rabbit polyclonal anti-TOM20 antibody was purchased from Santa Cruz Biotechnology (#sc-11415), and mouse monoclonal anti-GM130 antibody was from BD Biosciences (#610822). Alexa Fluor 555 conjugated anti-mouse IgG (#A31570), Alexa Fluor 555 conjugated anti-rabbit IgG (#A31572), Alexa Fluor 555 conjugated streptavidin (#S21381), Alexa Fluor 647 conjugated streptavidin (#S21374), and Alexa Fluor 647 conjugated wheat germ agglutinin (WGA) (#W32466) were from Thermo Fisher Scientific. Biotinylated WGA (#B-1025) was from Vector Laboratories. DyLight 549 (#S000-42) and DyLight 649 (#S000-43) conjugated streptavidins were from Rockland. HiLyte Fluor 555 (#AS-60666) and HiLyte Fluor 647 (#AS-60667) conjugated streptavidins were from Anaspec. iFluor 555 (#16989) and iFluor 647 (#16996) conjugated streptavidins were from AAT Bioquest. ATTO 647 (#AD647-61) streptavidin was from ATTO-TEC. SPICA dye-conjugated streptavidin was from Fujifilm Wako Chemicals. Biotin-SP AffiniPure goat anti-rabbit IgG (#111-065-144) was purchased from Jackson ImmunoResearch.
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