IPREM/LCABIE Laboratoire de Chimie Analytique Bio-inorganique et Environnement

Abstract

1 Introduction

Glycosyltransferases are accountable for the formation of specific glycan buildings which will have numerous natural outcomes. Lectins are sugar-binding proteins which have been used to detect various 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 specific developmental ranges.

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× (Top 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 elements distilled water, 2 elements 10 % paraformaldehyde and 1 half Embryo Fixation Stock Buffer. Make current sooner than each use.

3. Larval Fixation Decision: 4 % paraformaldehyde in 1× PBS. Make current 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 in opposition to 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).