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Step Four: Staining and
Observation
All
samples were fixed using 4% paraformaldehyde and placed
overnight in the incubator at
40oC.
All samples were washed in PBS and then were subjected to a
blocking agent for a half hour. For the primary antibody
stain, we used a 1:10 dilution of 3A10 and the blocking
agent. 3A10 is an IgG monoclonal antibody derived from mice,
that stains neurofilaments in chicks, rats, and mice. The
cultures were then left in the primary antibody for 24
hours.
For the secondary antibody,
we used a peroxidase-conjugated AffiniPure goat anti-mouse
IgG + IgM antibody. 1 mL of a dilution of 500 ( 2m IgG + IgM
: 1 mL PBS) was added to each well and left to sit for one
hour. The secondary antibody was then removed and 0.5 mL of
the substrate, Diaminobenzadine (DAB), (in a ratio of 9
parts buffer to 1 part DAB), was added to each well.
The substrate was left in and
monitored using a light microscope. As soon as the color
contrast was optimal, the substrate was removed and the
wells were filled with 2 mL of PBS.
Observation of the wells was
done using a light microscope at 10x, 20x, and 40x
magnification. Pictures were captured using ImageScope and
modified using Adobe Photoshop 5.5.
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