In certain experiments, the plasma membrane of the cells were stained with 2 µg/ml fluorescently labeled Wheat Germ Agglutinin (WGA, Life technologies) prior to fixation. Cells were rinsed with PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich, USA) for 15 min. Then the slides were pre-blocked with supermix (Tris-buffered saline, 0.25% gelatin, 0.5% Triton X-100) for 1 h at room temperature. Then the primary antibodies diluted in supermix were added to respective slides for overnight incubation at 4°C. After repeated washings with 1X PBS (Sodium perborate), they were incubated with secondary antibody (always 1∶400 dilutions in supermix when Alexa fluor is used, see Table 1 for details) for 1 h at room temperature. After several washing steps, DAPI (1∶2500 in PBS) was added for 10 min at room temperature. After washing, the slides were mounted in DTG media (with antifade (diazabicyclo (2.2.2) octane in 80% glycerol and 50 mM Tris pH 8.6) and photographed using a Zeiss AxioPlan 2 fluorescence microscope, connected to an AxioCamHRm camera. The micrographs were finally analyzed with Carl Zeiss AxioVision version 4.8 software.
Wheat germ agglutinin wga
Manufactured by Thermo Fisher Scientific
Sourced in United States
Wheat Germ Agglutinin (WGA) is a lectin derived from wheat germ. It is a protein that binds to and crosslinks specific carbohydrate moieties on the surface of cells.
Automatically generated - may contain errors
Lab products found in correlation
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Axiocam hrm camera, by Zeiss (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Axiovision version 4, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
X cite 120led boost high power led illumination system, by Excelitas (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Cellsens standard software, by Olympus (1 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Magnabind streptavidin beads, by Thermo Fisher Scientific (1 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
Synaptophysin, by Abcam (1 mentions)
Propidium iodide, by Biotium (1 mentions)
Calcein am, by Biotium (1 mentions)
Methacrylic anhydride, by Merck Group (1 mentions)
Connexin 43, by Abcam (1 mentions)
13 protocols using wheat germ agglutinin wga
1
Fluorescent Labeling of Cell Membranes
2
Visualization of Cellular siRNA Uptake
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For visualization of cellular siRNA uptake, 6 × 105 293TPSCA/ffLuc cells grown on a coverslip were incubated with scFv(AM1)-P-BAP-polyplexes loaded with Cy3-labelled siRNA for 24 h at 37 °C. After fixation of cells with 4% paraformaldehyde in PBS, cell membranes and nuclei were stained with Alexa Fluor 647 conjugated Wheat Germ Agglutinin (WGA, Life Technologies, Carlsbad, CA, USA) and Hoechst 33,342 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers protocols. The coverslips were placed upside down in a drop of mounting medium (Vector Laboratories) on a microscope slide. Cy3-labelled polyplexes conjugated with control scFv(MR1.1)-P-BAP, which cannot bind to 293TPSCA/ffLuc cells or without conjugation of scFv-P-BAP were included as negative controls. The images were captured by a confocal laser scanning microscope (Leica SP5, Leica, Wetzlar, Germany) and analyzed by Fiji software (ImageJ 1.51k, National Institute of Health).
3
Characterizing Skeletal Muscle Fiber Composition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Extracted gastrocnemii were immediately frozen in isopentane cooled in liquid nitrogen and stored at −80°C. Transversal sections of 5 µm thickness were cut at the midbelly with a cryostat. Sections were fixed for 10 min in PFA 4%, then blocked with 0.1% triton x‐100, 1% BSA in PBS before incubating with primary antibodies against fast/slow isoforms of myosin heavy chain and laminin (Abcam 91506, Abcam M8421, Santa Cruz 59854), followed by incubation with the corresponding secondary antibodies (Alexa‐488, Alexa‐568). For the electroporation of tibialis anterior with reporter plasmids, 8 μm cryosections were stained with AlexaFluor 555‐conjugated Wheat Germ Agglutinin (WGA, Thermo Fisher Scientific W32464) and DAPI. Pictures were taken with a fluorescent microscope and fiber areas were measured with ImageJ software (more than 500 fibers were analyzed per animal). The enzymatic activity of succinate dehydrogenase (SDH) was assessed on cryosections using the Succinic Dehydrogenase Stain (Bio‐Optica, 30–30114LY) according to manufacturer’s instructions.
4
Imaging Plasma EV Uptake in WI-38 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After a 2-hour coculture of WI-38 cells with PKH67-labeled plasma EVs (derived from 100 µl plasma) and the No EV control, the culture medium was discarded, and the cells on the plate were washed twice with 500 µl HBSS (Gibco). The cells were fixed with 100 µl 4% PFA for 15 minutes at 37°C, then washed three times with HBSS. WI-38 cell membranes were stained with Wheat Germ Agglutinin (WGA, ThermoFisher Scientific) and washed twice with HBSS. The cells were permeabilized with 0.2% Triton-X-100 (MilliporeSigma) for 5 minutes, followed by cell nuclei staining with DAPI (MilliporeSigma). The cells were washed twice with HBSS. Immunofluorescence images were captured on an Olympus IX70 Inverted Phase Contrast DIC Fluorescence Microscope accompanied with X-Cite 120LED Boost High-Power LED illumination System (EXCELITAS Technologies) and analyzed with cellSens Standard Software (Olympus).
5
Cardiac Remodeling Histopathology Analysis
6
Isolation and Characterization of Aged Yeast Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Old cells were obtained using the magnetic beads biotin-streptavidin system according to established protocols [35 (link)]. Cells were grown to exponential phase at 30 °C, approximately 3 × 107 cells were harvested, washed in PBS and then labelled with EZ link Sulfo-NHS-LC biotin (Thermo Scientific) at a final concentration of 0.5 mg/mL in 1 ml. Excess biotin was washed away, and cells were resuspended in 1L growth medium and cultured for about 15 h overnight at 30 °C, using an orbital shaker (180 rpm). When the cultures had reached an OD600 of about 0.5, cells were washed with PBS and incubated with 0.05 mg/ml MagnaBind streptavidin beads (Thermo Scientific). Biotin-labelled cells were then isolated using a magnetic sorter and continuous washes with PBS containing 0.5% glucose. Cells were resuspended in a 7 ml growth medium and used directly for subsequent experiments. The median age of the old cells was determined by counting bud scars in z-stack images upon staining cells with 10 µg/mL Wheat Germ Agglutinin (WGA, Thermo Scientific).
7
Immunofluorescence Visualization of Proteins
8
Immunolabeling of Aquaporin Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A polyclonal affinity purified anti-AQP0 antibody, directed against the last 17 amino acids of the C-terminus of the human protein, was obtained from Alpha Diagnostic International (San Antonio, TX, USA, catalogue number AQP01-A). An affinity purified polyclonal anti-AQP5 antibody, directed against the last 17 amino acids of the C-terminus of the rat protein, was obtained from Merck Millipore (Darmstadt, Germany, catalogue number AB15858). A rabbit polyclonal antibody directed against the last 19 amino acids of the C-terminus of the rat AQP1 protein was purchased from Alpha Diagnostic International. Secondary antibodies (goat anti-rabbit Alexa 488) and wheat germ agglutinin (WGA) conjugated to a fluorophore (WGA–Alexa 594) for labeling of the cell membrane were obtained from Thermo Fisher Scientific (Waltham, MA, USA). For labeling of the epithelial and fiber cell nuclei , DAPI">DAPI was obtained from Sigma-Aldrich (St Louis, MO, USA). Phosphate-buffered saline (PBS) was prepared fresh from PBS tablets. Lenses were organ cultured in artificial aqueous humor (AAH) that consisted of (in mM) 125 NaCl, 0.5 MgCl2, 4.5 KCL, 10 NaHCO3, 2 CaCl2, 5 glucose, 10 sucrose, 10 HEPES (pH 7.4), and 300 mOsml/L. Tropicamide and pilocarpine were used at 1:5 dilution prepared in AAH buffer from 1% w/v eye drops. Unless otherwise stated, all other chemicals were from Sigma-Aldrich.
9
Porcine Skin Gelatin Hydrogel Fabrication
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gelatin from porcine skin (Type A, SLCC7838), methacrylic anhydride (MA), Irgacure (2‐Hydroxy‐4′‐(2‐hydroxye‐thoxy)‐2‐methylpropiophenone), and PBS were obtained from Sigma‐Aldrich (Wisconsin, U.S.A.). AlamarBlue and qPCR kits were purchased from Bio‐Rad (Hercules, U.S.A). Carbopol ETD 2020 polymer was purchased from Lubrizol (Wickliffe, U.S.A.). Calcein AM and propidium iodide (PI) were obtained from Biotium (Fremont, U.S.A.). HUVEC culture medium (VascuLife VEGF) was purchased from Lifeline Cell Technology (Oceanside, U.S.A.). Conjugated F‐actin and wheat germ agglutinin (WGA) were purchased from ThermoFisher, USA; CD31, Connexin 43, Phox2B and Synaptophysin antibodies were purchased from Abcam, USA.
10
Immunofluorescence Staining of Fixed Cells
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!