ECM Macromolecules: Rotary Shadowing and Scanning Transmission Electron Microscopy - Methods in Molecular Biology: Extracellular Matrix Protocols - page 113

Fig. 1. Rotary shadowed fetal bovine dermal microfibrils. (A) Type VI collagen
microfibrils (white arrow); fibrillin-containing microfibril (black arrow). (B) “Beads
on a string” morphology of fibrillin-containing microfibrils. At higher magnifications
the granularity of the background becomes apparent. Bar = 200 nm.
are complex multicomponent polymers which have proved amenable to STEM-
mass mapping techniques. Microfibrils incubated in the presence of calcium
chelating agents or with raised calcium concentrations ( Fig. 3 ) demonstrate
that their morphology is exquisitely sensitive to calcium (4) .
Multicomponent assemblies may be probed for constituent molecules using
antibody binding. STEM analysis can detect the presence of antibodies by mass
alone eliminating the need for an intrinsically inefficient secondary antibody-
immungold complex binding step. The same techniques can be employed to
quantify specific enzyme susceptibilities and binding affinities between native
assemblies and other matrix components. Pathological and developmental pro-
cesses can be investigated using STEM-mass mapping techniques to probe for
the presence of distinct associated macromolecules. The number and orienta-
tion of molecules in a complex can also be determined using this technique.
The composition of multicomponent assemblies can also be probed using
specific enzyme digests and STEM. Mass loss following enzyme digestions
was shown following incubation of fibrillin microfibrils with chondroitinase
ABC lyase (5) . These techniques are applicable to macromolecular assemblies
ranging in mass from 10 kDa/nm to over 9000 kDa/nm ( Fig. 4 ). The protocols
in this chapter cover the isolation of fibrillar collagens and microfibrils, the
production of carbon-coated grids, rotary shadowing of grids and mica, and the
techniques of STEM and associated image analysis.
2. Materials
2.1. Extraction and Isolation of Collagen Fibrils and Microfibrils
All reagents are obtainable from BDH or Sigma except where stated. All
solutions use double-distilled ultrapure water and may be stored at room tem-
perature, unless otherwise stated.