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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.

©Cebra-Thomas, 2000

Last Modified: 5 May 2000

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