Biology Reference
In-Depth Information
2.
Homogenization for 45 s using a Dounce homogenizer is usually sufficient to
enable extraction of macromolecules from young embryonic or fetal tissue.
Additional ultrasonic treatment and repeated Dounce homogenization may be
required for older tissues. When ultrasonicating tissue samples, care must be
taken to avoid heat denaturation.
3.
The digestion rate varies with the tissue source. Skin from bovine fetuses will
digest completely within 2-4 h at room temperature. Aorta and adult bovine skin
require an overnight digestion.
4.
Bacterial collagenase resistant type VI collagen microfibrils are copurified with
fibrillin containing microfibrils by this technique. Dilute suspensions of
microfibrils may be concentrated by centrifugation using microcon or
centricon concentrators of 100 kDa cutoff (Amersham Life Sciences,
Buckinghamshire, UK).
5.
Using a lathe, cut a 6-mm diameter carbon rod to form a tip approximately 0.8 mm
in diameter and 1 mm in length. Form the tip by progressively cutting concentric
circles of decreasing diameter (
Fig. 5
).
Cut a 2-cm
2
sheet of mica and trim 1 mm from each edge. Using the side of a pair
of forceps flatten one edge of the mica. Gently insert one blade of the forceps into
the flattened side until the mica cleaves.
6.
7.
Where no monitor is available, a carbon-coating apparatus of the dimensions
detailed in
Fig. 5
should produce films within the required film thickness range.
Films should be self supporting, but not too thick. A thick carbon film signifi-
cantly reduces the signal-to-noise ratio for low-mass samples such as microfibrils
and collagen fibril tips.
8.
An absorbent newspaper is ideal. Avoid areas of ink, particularly color printing.
9.
The sample drop should spread over the surface of the grid. If problems are
encountered, gently circle the pipette over the grid to spread the sample evenly.
Adsorption times of greater than 30 s increase background contamination.
10.
Samples may be freeze-dried as an alternative to air-drying.
11.
The shadowing angle may be varied up to 10
°
. Larger angles improve image
contrast but result in increased grain size.
12.
Use welders goggles or glasses of a similar opacity.
13.
Combined STEM/TEM instruments (capable of producing a 2-3-nm spot in
STEM mode and equipped with efficient photomultiplier/scintillator ADF detec-
tors) are available from JEOL Ltd. (Tokyo, Japan), Philips Electron Optics B.V.
(Eindhoven, Netherlands), and Carl Zeiss Ltd. (Oberkochen, Germany).
14.
Data acquisition systems (ES Vision) are available from Emispec Systems Inc.
(Arizona, USA).
15.
Our laboratory uses the Semper 6 image analysis language (Synoptics, Cam-
bridge, UK). Semper 6 is no longer available commercially and has been replaced
by the RAD tool (Visual Basic, Visual C
++
and Delphi 3) Image Objects from the
same company. Other commercial and public domain packages are also available.
16.
Typical magnifications; TMV =
×
20,000, microfibrils =
×
80,000, fibrillar col-
lagen =
×
25,000/
×
50,000.
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