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Lab Standard Operating Procedure (SOP) for Immunohistochemical Staining
on Cell Cultures
Materials
Literature
1.  Using
a disposable plastic pipet with a fine tip, remove the solution
from the wells and apply 10%NBF for 10 min. to cover cells.
2. Remove the 10%NBF and wash 3-5X with Tris with Tween buffer (Dako
S3306) tipping the well plate and using the fine tip of the pipet
trying not to disturb the cells.
3. Apply 0.03% H202 with sodium azide (Dako) for 5 min. then remove
and follow with 3 buffer washes.
4. After removing the last buffer wash apply Protein Block (Dako
X0909) for 10 min. incubation
5. Remove the protein block with pipet and apply primary antibody
for overnight incubation.
6. The following morning remove the primary antibody and wash the
cells with Tris w. Tween buffer X3 times
7. Apply the secondary antibody or Link (Dako LSAB+ kit K0690) for
30 min.
8. Remove secondary and wash with buffer 2-3 times.
9. Apply the Streptavadin (Dako LSAB+ kit K0690) for 30 min.
10. Remove Streptavidin and wash cells 3-5 times with Tris buffer
with Tween.
11. Apply DAB (Dako, K3466) and develop for 5-10 min.
12. Rinse cells with distilled water X5 times.
Last
GLP modification 9/13/04:
Jennifer
Casati
James R. Turk
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