Leica Microsystems EM ICE Application Note

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Application Note

SUPERIOR ULTRASTRUCTURAL PRESERVATION AND

STRUCTURAL CONTRAST IN DROSOPHILA TISSUE

related instruments: EM ICE

SUPERIOR ULTRASTRUCTURAL PRESERVATION

AND STRUCTURAL CONTRAST IN DROSOPHILA

TISSUE

Application Note for High Pressure Freezer EM ICE

Zulfeqhar A. Syed

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

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 nger-like protrusions termed as gastric caeca, that are

responsible for active secretion of most digestive enzymes

(1,2,3)

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 at 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 inltration was per-

formed by incubating samples in increasing concentrations of Lowicryl HM20 resin (25%, 50%, 75%) in acetone with nal 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 an 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.

References:

(1)

Demerec, M. Biology of Drosophila., (1950) New York, John Wiley and Sons Inc.; London, Chapman and Hall Ltd.

(2)

Bodenstein, D. The

post-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)

LNT Application Note - DROSOPHILA TISSUE 3

500 nm

200 nm

5 µm

The top panel shows an overview of Drosophila gastric caeca and a mitochondrion with well-dened cristae and well-preserved surrounding

membranes. The lower panel shows a smooth septate junction, which forms regions of close membrane contact that extend over a large part

of the basolateral membrane in the endoderm-derived epithelia.

Micrographs courtesy of Drs Syed (NIH/NIDCR) and Bleck (NIH/NHLBI)

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