Biomedical Engineering Reference
In-Depth Information
Fig. 4.1.
Stamp (
A
) and slide (
B
) with two times of six sample wells (1-6)
and larger central well (
C
)
C
0.05% Coomassie Brilliant Blue G250 (w/v) or 0.1% Amido
Black 10 B in B
PBS
Melt Reagent A in a water bath or in a microwave oven. Dilute some
milliliters of the molten solution with hot water to get 0.5% agar or
agarose. Pour this solution onto a carefully defatted slide (3
7 cm)
and dry in a stream of warm air. Store a stock of these pre-coated
slides dustless; they are stable at RT for months.
Pipet 4 ml of the hot molten Soln. A onto the covered slides lay-
ing on a horizontally leveled table. After gelation, punch wells using
a stamp as shown in Fig. 4.1. Suck out carefully residual material
from the wells using a Pasteur pipette connected to a pump.
×
4.8.3 Immunodiffusion
Depending on the question, fill the central well either with the
antigen solution or with antiserum. Place the antigen(s) or antisera
or antiserum dilutions into the peripheral wells. If the wells are not
filled completely, adjust with PBS.
Place the slides horizontally in a humid chamber, e.g., Petri
dishes containing wet filter paper, and store in a refrigerator at
least for 24 h,betterfor48-72h.
4.8.4 Visualization of the Precipitin Lines
When diffusion is complete, strong precipitation lines are visible
when the slide is viewed obliquely against a black background.
Weak lines are visualized by staining.
