Biology Reference
In-Depth Information
2. Materials
2.1. Protein and Carbohydrate Labeling
1.
Protein labeling buffer, 0.2
M
: NaHCO
3
(1.48 g/100 mL), Na
2
CO
3
(0.24 g/100 mL),
pH 9.0. Filter 0.2-mm before use. Should be prepared fresh, keeps at 4
°
C for 2 wk.
2.
Phosphate-buffered saline (PBS): 8 g NaCl, 0.2 g KCl, 1.44 g Na
2
HPO
4
. 2H
2
O,
0.2 g KH
2
PO
4
, made up to 1 L, pH 7.4. Filter 0.2-
µ
m before use, keep at 4
°
C.
3.
Carbohydrate (hyaluronan) labeling buffer, 0.2
M
: Na
2
B
4
O
7
.10H
2
O (0.572 g/
100 mL), boric acid (0.866 g/100 mL), pH. 8.0. Filter 0.2-
µ
m before use.
4.
FITC.
5.
Hyaluronan (HA).
6.
CNBr.
7.
Hi-trap columns.
8.
Fluoresceinamine.
9.
Centriplus (Amicon, Danvers, MA).
10.
Dialysis tubing.
2.2. Photobleaching Experiments
1.
Upright confocal laser scanner and fluorescence microscope: the authors use a
100 mW Argon-Ion laser (
see
Note 1
) with a MRC-1000 scanner (Bio-Rad,
Hemel Hempstead, UK) attached to a Optiphot microscope (Nikon [Membrane
filtration Products, San Antonio, TX]) (
Fig. 3
). The chosen scan head needs to
include a motorized intensity filter changer and a fluorescein compatible filter
set. With the MRC-1000, data acquisition and analysis are controlled by a simple
programming language (MPL) for which the authors have written appropriate
“macro” procedures. The majority of confocal microscopes include similar
facilities for user application development.
2.
Microscope objectives of numerical aperture <0.6 (magnification:
20). A
low numerical aperture is most appropriate, as it gives a more uniform and paral-
lel bleach volume, allowing the results to be calculated using a 2-dimensional
analysis
(15)
.
×
10,
×
3.
Temperature-controlled microscope stage capable of operating between 5 and
60
°
C at
±
0.1
°
C, for example Linkam Instruments PE 60 (Linkam Ltd, UK).
4.
Cavity microscope slides, 12-mm cavity diameter, 30-
L volume (Scientific Lab
Supplies, Nottingham, UK). Slides should be scrupulously cleaned and rinsed
with deionized water before use.
µ
3. Methods
3.1. Sample Preparation and Fluorescence Labeling
Glycosaminoglycans, including chondroitin sulphate and hyaluronan, are
available commercially (e.g., Sigma Corp., St. Louis, MO, Seikagaku Corp.,
Tokyo, Japan) from several tissue sources (e.g., bacteria, cock's comb, umbili-
cal cord), although the molecular weight and degree of protein contamination
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