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Superior Ultrastructural Preservation and Structural Contrast in Drosophila Tissue

Application Note for High Pressure Freezer EM ICE

Optimal structural preservation of tissue can only be obtained by high pressure freezing. However, preparing the samples in optimal conditions is challenging. This article explains in detail how to dissect, high pressure freeze and freeze substitute Droshophila tissue sample to obtain high structural integrity for subsequent electron microscopic analysis.

Zulfeqhar A. Syed1 and Christopher K. E. Bleck | Developmental Glycobiology Section, National Institute of Dental and Craniofacial Research, National Instiutes of Health,

Bethesda, MD, USA | Electron Microscopy Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

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Introduction

The Drosophila larval gut is a simple epithelium which is divided into three distinct compartments, the foregut, midgut, and hindgut. The proventriculus is a bulb shaped organ situated at the junction of foregut and the midgut, and functions as a valve controlling the entry of food into the midgut. The posterior end of the proventriculus, there are four long finger-like protrusions termed as gastric caeca, that are responsible for active secretion of most digestive enzymes(1,2,3).

Procedure

A portion of anterior midgut from Drosophila third instar larva consisting of the proventriculus and gastric caeca was dissected in Schneider’s medium and transferred to a type-A planchet, cavity depth 0.2 mm. Excess of Schneider’s medium was carefully removed and immediately 1 μl of 20% BSA/PBS was pipetted and uniformly distributed. After inspecting sample orientation, a Type-B planchet was placed on top with flat surface down to seal the assembly.

The assembled specimen chamber was frozen using a Leica EM ICE high-pressure freezing system. The frozen samples were transferred to cryovials in liquid nitrogen vapour and transferred to pre-cooled (-90°C) freeze substitution unit (Leica EM AFS). Freeze substitution was performed using a mixture of 0.12% aqueous uranyl acetate in anhydrous acetone using the following program: -90°C for 45 hrs followed by slow warming from -90°C to -50°C (15°C/hr). At -50°C freeze substitution solution was removed and the samples were washed 3 times for 10 mins with acetone. Resin infiltration was performed by incubating samples in increasing concentrations of Lowicryl HM20 resin (25%, 50%, 75%) in acetone with final 3 incubations with 100% resin lasting for 48 hrs. The samples were gradually warmed from -50°C to 24°C (5°C/hr) and UV polymerized over a period of 48 hrs. Ultrathin sections (50-60 nm) were cut on a Leica EM UC7 ultramicrotome and postained with 2% of aqueous uranyl acetate for 10 mins and lead citrate for 2 mins. Digital micrographs were acquired on JEOL JEM 1200 EXII operating at 80kV and equipped with bottom mounted AMT XR-60 digital camera.

Results

References

  1. Demerec, M. Biology of Drosophila., (1950) New York, John Wiley and Sons Inc.; London, Chapman and Hall Ltd.
  2. Bodenstein, D. Thepost-embryonic development of Drosophila., (1950), pp.275-367, Hafner, New York;
  3. The embryonic development of Drosophila melanogaster:By J. A. Campos-Ortega and V. Hartenstein. New York: Springer-Verlag. (1985)