Biology Reference
In-Depth Information
2.2. Ultrastructural Investigations
Materials and preparative equipment for electron microscopy may be pur-
chased from Agar Scientifc.
1.
Carbon rods (6-mm diameter).
2.
Braided carbon fiber.
3.
Electron microscope grids (400 mesh copper or nickel).
4.
Mica sheets (25
×
25 mm, 0.15-mm thick).
5.
Tungsten wire (0.5-mm diameter).
6.
Platinum wire (0.1-mm diameter).
7.
Diffraction grating (2160 lines/mm).
8.
Fine tweezers with clamping ring.
9.
0.2
M
ammonium acetate (pH 6.0).
10.
Liquid nitrogen.
11.
Freeze-drying table (0.5-cm thick copper sheet supported by legs with a cen-
tral handle).
12.
High vacuum coating unit: large bell jar (30-cm diameter), rotating table with
variable speed control (50-200 rpm), power supply providing 10 V/100 A.
3. Methods
3.1. Extraction and Isolation of Fibrillar Collagens
1.
Dissect 0.5 g of the tissue sample (tendon, skin, cornea). Wash in fibrillar collagen
extraction buffer (
see
Note 1
) and cut into 1-mm
2
pieces.
2.
Homogenize 0.5 g of the tissue sample in 0.5 mL fibrillar collagen extraction
buffer using a hand-held Dounce homogenizer for 45 s (
see
Note 2
).
3.
Fibril-rich solutions are stable at room temperature for 2 wk.
3.2. Extraction and Isolation of Microfibrils
Dissect 1 g of tissue (skin, aorta, nuchal ligament, ciliary zonules) into 1 mm
3
pieces.
1.
2.
Incubate the tissue fragments with 1-2 mL active collagenase buffer on a rotary
mixer until no visible fragments of tissue remain (
see
Note 3
).
3.
Following digestion, centrifuge the samples at 7840
g
for 10-30 min. The cen-
trifugation step removes remaining aggregates (which could block the column).
4.
Chromatograph the supernatant on the preequilibrated Sepharose CL-2B gel fil-
tration column at a flow rate of 0.2 mL/min and a fraction size of 1 mL. High-
M
r
material in the excluded volume (
V
o
) includes fibrillin-containing and type VI
collagen microfibrils (
see
Note 4
).
5.
Isolated microfibrils suspended in column buffer may be stored at 4
C for 7 d
with no changes in gross morphology or mass distribution as determined by
RS-TEM and STEM mass mapping techniques.
°
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