Hearts were excised and immediately placed in a 10% potassium chloride solution to guarantee that the hearts were arrested in diastole. Then, the hearts were fixed for more than 24 hours in 10% formalin, dehydrated, and embedded in paraffin. Subsequently, the hearts were transversely sectioned at 5 μm when the section is close to the left and right ventricles. Sections of each sample were stained with hematoxylin–eosin (HE) and picrosirius red following standard procedures. Furthermore, sections were stained with fluorescein isothiocyanate–conjugated wheat germ agglutinin (WGA, Invitrogen Corp) to confirm the results observed in HE staining (WGA for cell membrane and DAPI for nuclei). We measured the cross‐sectional area (HE) and LV collagen volume (picrosirius red) by Image‐Pro Plus 6.0. More than 100 LV cardiomyocytes’ cross‐sectional areas and more than 25 fields were measured in each group.
Wheat germ agglutinin (wga)
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
The WGA is a lab equipment product that performs whole genome amplification. It is designed to generate high-quality, representative amplification of genomic DNA from small samples or low-input DNA.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (11 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (7 mentions)
Hoechst 33342, by Thermo Fisher Scientific (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
Hoechst, by Thermo Fisher Scientific (4 mentions)
Triton x 100, by Merck Group (3 mentions)
Bx53 microscope, by Olympus (3 mentions)
Masson s trichrome, by Merck Group (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Slowfade, by Thermo Fisher Scientific (3 mentions)
Lsm 510, by Zeiss (3 mentions)
Bodipy, by Thermo Fisher Scientific (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Cellomics arrayscan vti, by Thermo Fisher Scientific (2 mentions)
Oregon green 488 conjugate, by Thermo Fisher Scientific (2 mentions)
Egf af555, by Thermo Fisher Scientific (2 mentions)
Anti cd31, by Abcam (2 mentions)
Ab125212, by Abcam (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
187 protocols using wheat germ agglutinin (wga)
1
Cardiac Histological Analysis Protocol
2
Immunofluorescence Staining of Heart Vessels
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Immunofluorescence staining was performed as described previously to analyze number of vessels19 (link). Histological sections of the heart were incubated with monoclonal rat anti-CD31 (Dianova, Hamburg, Germany) or rabbit polyclonal anti-alpha smooth muscle actin antibodies (Abcam,Cambridge, UK) over night at 4 °C. For WGA staining histological sections were incubated at room temperature with WGA (Invitrogen, Carlsbad, CA) for 15 min. Five areas per sample were analyzed. Sections were examined with a Zeiss Axioplan 2 (Zeiss, Oberkochen, Germany) and a Leica SP5 confocal microscope (Leica, Wetzlar, Germany). Images were analyzed using ImageJ software (NIH, Bethesda, MD, USA) and processed with Adobe Photoshop CS5.1 for Mac (Adobe Systems Inc., San Jose, CA, USA)30 .
3
Bacterial Aggregate Visualization in Synovial Fluid
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Bacteria were stained with BacLight Green (Thermo Fisher Scientific, Waltham, MA) prior to inoculation of synovial fluid; after overnight culture in synovial fluid, macroscopic aggregates were gently removed from synovial fluid and washed three times with phosphate buffered saline (PBS). Aggregates were suspended in PBS and stained with wheat germ agglutinin (WGA, Thermo Fisher Scientific, Waltham, MA; 20μg/mL) for 15 min at room temperature in the dark. Supernatant containing WGA was removed and aggregates were stained with 1mL undiluted SYPRO (FilmTracer SYPRO Ruby Biofilm Matrix Stain, Thermo Fisher Scientific, Waltham, MA) for 30 min at room temperature in the dark. Thereafter, stain was removed and aggregates were washed three times with PBS before fixation in 2% paraformaldehyde. Aggregates were kept at 4°C until imaging. Imaging was performed using a Leica SP5 Confocal/Multiphoton Microscope at the PennVet Imaging Core.
4
Visualization of Glycans and Lipids in Tissue Sections
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For WGA staining, the paraffin sections were dewaxed, rehydrated, and stained with 5 μg/mL WGA (Thermo Fisher Scientific, cat W11261, USA) in PBS for 30 min at room temperature. Then, the sections were washed with PBS 3 times and observed with a FluoView™ FV3000 confocal microscope (Olympus, Japan).
For BODIPY staining, the mouse hearts were fixed in 4% PFA, dehydrated in 30% sucrose, embedded in OCT and sectioned into 8 μm slices. Frozen sections were thawed at room temperature and stained with 2 μM BODIPY (Thermo Fisher Scientific, cat D3922, USA) for 15–20 min at 37 °C. Then, the sections were washed with PBS 3 times and observed with a FluoView™ FV3000 confocal microscope (Olympus, Japan).
For BODIPY staining, the mouse hearts were fixed in 4% PFA, dehydrated in 30% sucrose, embedded in OCT and sectioned into 8 μm slices. Frozen sections were thawed at room temperature and stained with 2 μM BODIPY (Thermo Fisher Scientific, cat D3922, USA) for 15–20 min at 37 °C. Then, the sections were washed with PBS 3 times and observed with a FluoView™ FV3000 confocal microscope (Olympus, Japan).
5
Microscopic Examination of Plant Tissue
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Stromata and substromata samples were cut in approx. 0.1 cm cross sections by hand with a scalpel blade. Leaf sheath and blade samples were cut in 2 cm long subsamples. All samples were stained with Wheat Germ Agglutinin, Alexa Fluor™ 488 Conjugate (ThermoFisher Scientific, MA, USA) and Aniline Blue diammonium salt (C37H32N5O9S3) and eventually with propidium iodide (both: Sigma-Aldrich Chemie, Traufkirchen, Germany) as described by Becker et al. (2018 (link)). Samples were transferred to microscopic slides and embedded in staining solution for 10 min before microscopic examination. Confocal laser scanning microscopy (CLSM) was done using a Leica TCS SP8 as described in Becker et al. (2018 (link)).
6
Macrophage Trafficking Dynamics Assay
7
Hsp90 Localization in MCF10DCIS.com Cells
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MCF10DCIS.com cells were resuspended in DMEM/F12 medium supplemented with 5% horse serum, seeded on glass-bottom dishes (MatTek, Ashland, MA, USA) and incubated overnight at 37 °C. Cells were washed with PBS twice and incubated with anti-Hsp90 mAb (AC88, 1 µg/mL, Abcam) for 30 min on ice. Then, cells were washed with PBS and fixed with 10% neutral buffered formalin for 15 min at 37 °C. After the removal of formalin, the cells were then incubated with secondary antibody (1:500 dilution, AF568-conjugated goat anti-mouse IgG, Cat# A-11004, Life Technologies, Carlsbad, CA, USA), followed by Wheat Germ Agglutinin (WGA) Alexa Fluor 488 conjugate membrane staining dye (5 µg/mL, Cat# W11261, ThermoFisher) for 20 min and DAPI (Cat# 422801, BioLegend, San Diego, CA, USA) for 10 min at room temperature. After being washed with PBS, cells in the dish were observed using a ZEISS LSM880 confocal microscope (Carl Zeiss AG., Oberkochen, Germany).
8
Histological Evaluation of Cardiac Hypertrophy
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Hearts were arrested by perfusion of KB buffer (20 mmol/L KCl, 10 mmol/L KH2PO4, 70 mmol/L K+‐glutamate, 1 mmol/L MgCl2, 25 mmol/L glucose, 20 mmol/L taurine, 0.5 mmol/L EGTA, 10 mmol/L HEPES, 0.1% albumin, and pH adjusted to 7.4 with KOH) and were perfusion fixed with 4% paraformaldehyde at constant pressure. Hearts were fixed an additional 2 days at 4°C with rotation, then processed and embedded in paraffin by standard methods and sectioned at 4 μm. To assess cardiomyocyte hypertrophy, sections were stained with Wheat Germ Agglutinin (catalog number W6748, ThermoFisher Scientific) and imaged on a widefield microscope. DAPI was used to identify nuclei. Cardiomyocyte cross‐sectional areas were measured in cardiomyocytes with a centrally located nuclei using FIJI software27 Fibrosis was measured by staining with Masson's trichrome, and cardiac collagen content was determined as the percentage of collagen in sections of the left ventricle. Cardiac apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end‐labeling (TUNEL) positivity. LV sections were subjected to the ApopTag Plus Peroxidase kit (MilliporeSigma, Inc) according to the manufacturer's instructions and costained with DAPI for nucleus identification. TUNEL positivity was calculated by dividing the total number of TUNEL‐positive cells by the number of nuclei.
9
Quantifying Cardiac Morphology and Vasculature
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After euthanasia, the heart tissue was excised, flushed with 1 mol/L KCl solution, and fixed in 10% formalin, embedded in paraffin, and sectioned at 4 µm. Heart cross sections were stained with 4′,6-diamidino-2-phenylindole (Invitrogen) and wheat germ agglutinin (ThermoFisher) for quantification of cardiomyocyte cross-sectional area. In addition, sections were stained with isolectin B4 (Vector Laboratories) to assess capillary density and with Sirius red for fibrosis. Quantitative measurements were determined using Nikon Elements software.
10
Filipin Labeling of Unesterified Cholesterol
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After treatment, cell membranes were labeled with wheat germ agglutinin® (Thermo Fisher). Cells were fixed in 4% PFA for 20 min, washed 3× in PBS, and 1× in glycine. Unesterified cholesterol was labeled with filipin labeling solution for 2 h. Filipin labeling solution: 10% FBS + 0.4% DMSO+ 0.03 mg/ml (tissue) or 0.1 mg/ml (cells) filipin. Slides were washed 3× with PBS and mounted with ProLong® Gold (Thermo Fisher) [25 (link)].
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