fitc conjugated vicia villosa

FITC Conjugated Vicia villosa Lectin

Abstract

Glycans are the outcomes of the coordinated actions of glycosyltransferases accountable for explicit sugar additions. Glycans present on proteins can have an effect on protein stability, transport, function, and recognition, and thus can have profound outcomes on cell–cell interactions, adhesion, and signaling events occurring all through eukaryotic enchancment. Lectin staining provides a fantastic software to detect the spatial distribution of explicit glycans in creating tissues in situ. Proper right here we describe a method to detect numerous glycans present in creating Drosophila tissues and organs using fluorescently labeled lectins.

Key phrases: Glycosylation, Glycans, Lectins, Confocal microscopy, Drosophila, Development

1 Introduction

Glycosyltransferases are accountable for the formation of explicit glycan buildings that will have numerous natural outcomes. Lectins are sugar-binding proteins which have been used to detect numerous glycan buildings present all through eukaryotic enchancment [1, 2]. In an effort to seek out out the spatial distribution of glycans all through Drosophila enchancment, we have stained Drosophila embryos and tissues with fluorescently labeled lectins and carried out confocal microscopy [3]. This method is acceptable with whole-mount staining, allowing one to image glycan expression in a 3D embryo or tissue at explicit developmental ranges.

fitc conjugated vicia villosa
fitc conjugated vicia villosa

2 Provides

Put collectively all choices using distilled water. Strictly adjust to all storage and waste disposal procedures.

  1. Bovine serum albumin (BSA): Albumin bovine fraction V (MP Biomedicals LLC., Solon, OH, USA). Retailer at 4 °C.

  2. Dechorionation reply: Germicidal bleach, energetic ingredient is 6.15 % sodium hypochlorite (Clorox Expert Merchandise Agency, Oakland, CA, USA). Retailer at room temperature.

  3. Heptane (Sigma-Aldrich, St. Louis, MO, USA). Retailer at room temperature in flame resistant cabinet.

  4. Glycerol (Life Utilized sciences, Carlsbad, CA, USA). Make 70 % glycerol reply with 1× PBS. Retailer at room temperature.

  5. Methanol: Methyl alcohol, anhydrous (Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA.). Retailer at room temperature in flame resistant cabinet.

  6. Paraformaldehyde: 10 % stock reply, EM grade, methanol and RNase free (Electron Microscopy Science, Hatfield, PA, USA). Retailer at room temperature.

  7. Phosphate Buffered Saline (PBS), 10× (Prime quality Natural, Inc., Gaithersburg, MD, USA). Dilute to 1× with distilled water. Retailer at room temperature.

  8. Triton X-100 (Sigma-Aldrich). Retailer at room temperature.

  1. Embryo Fixation Stock Buffer: 800 mM KCl, 200 mM NaCl, 150 mM PIPES (1,4-Piperazinediethanesulfonic acid, Sigma-Aldrich), and 20 mM EGTA (ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid, Sigma-Aldrich) in 400 mL. Alter pH to 7.4 with 5 N NaOH (see Remember 1) and convey to closing amount of 500 mL. Autoclave to sterilize. Retailer at 4 °C.

  2. Embryo Fixation Working Decision: Combine 2 parts distilled water, 2 parts 10 % paraformaldehyde and 1 half Embryo Fixation Stock Buffer. Make latest sooner than each use.

  3. Larval Fixation Decision: 4 % paraformaldehyde in 1× PBS. Make latest sooner than each use.

  4. Lectins: Fluorescent lectins purchased from EY Laboratories (San Mateo, CA, USA) included tetramethylrhodamine isothiocyanate (TRITC)-conjugated Canavalia ensiformis (Con A); TRITC-conjugated Dolichos biflorus (DBA); fluorescein isothiocyanate (FITC)-conjugated Artocarpus integrifolia (Jacalin); FITC-conjugated Vicia villosa (VVA); and FITC-conjugated Triticum vulgare (WGA). Lectins purchased from Molecular Probes (Eugene, OR, USA) included Alexa Fluor 488-conjugated Arachis hypogaea (PNA) and Alexa Fluor 568-conjugated Glycine max (SBA). FITC-conjugated Helix pomatia (HPA) (Sigma-Aldrich). Lectins had been reconstituted at 1 mg/mL in distilled water and saved as 20 µL aliquots at −20 °C (see Remember 2). All fluorescent lectins must be protected towards gentle.

  5. Sugars (Sigma-Aldrich) for inhibition of lectin binding: N-Acetyl-d-galactosamine (GalNAc), for inhibition of HPA, DBA, SBA and VVA (retailer at 4 °C); N-Acetyl-d-glucosamine (GlcNAc) for inhibition of WGA (retailer at −20 °C); d-(+)-Galactose (Gal) for inhibition of Jacalin and PNA (retailer at room temperature); d-(+)-Mannose (Man) for inhibition of Con A (retailer at room temperature). Make 1 M stock reply of each sugar in 1× PBS. Retailer stock choices at −20 °C.

  6. Hoechst 33342 nuclear counterstain, 10 mg/mL stock reply (Molecular Probes, Eugene, OR, USA). Retailer at 4 °C.

  7. Mounting Decision: 2 % 1,4-Diazabicyclo[2.2.2]octane (Dabco, Sigma-Aldrich) in 70 % glycerol. Retailer at −20 °C.

  8. Glass slides: 75 × 25 mm Superfrost/Plus Microslides (Daigger, Vernon Hills, IL, USA).

  9. Cowl slips: 24 × 50mm microscope cowl glass, No. 1 (Thermo Scientific, Portsmouth, NH, USA).

  10. Cell strainer, 70 µm nylon mesh (BD Biosciences, San Jose, CA, USA).

  11. Pink sable paintbrush, dimension 2 (Thomas Scientific, Swedesboro, NJ, USA).

  12. 7 mL scintillation vials with unattached cap (Daigger).

  13. GyroMini™ Nutating Mixer (Labnet Worldwide, Woodbridge, NJ, USA).

  14. Pyrex Spot Test Plates, 9 properly, 85 × 100 mm (Thomas Scientific).

  15. Dissecting Dish with Plastic Dish (Electron Microscopy Science).

 

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