|
Staining for First Antigen
|
|
1. Preparation of tissue. Fix sections with the appropriate fixative for the antigen under study (Please see Note 1).
|
|
2. Air dry sections.
|
|
3. Wash sections 2 x 2 minutes in buffer (PBS).
|
|
4. Avidin/biotin blocking step. Perform Avidin/Biotin blocking if required (Avidin/Biotin Blocking Kit, Cat. No. SP-2001). Incubate sections with Avidin Solution for 15 minutes. Rinse briefly with buffer, then incubate in the Biotin Solution for 15 minutes. Wash sections 2 x 2 minutes in buffer. This blocking step may be eliminated if suitable controls have determined this step to be unnecessary.
|
|
5. Protein blocking step. Incubate sections for 20 minutes with buffer containing 5% normal blocking serum (NS) which was prepared from the species in which the secondary antibody is made.
|
|
6. Blot excess serum from sections.
|
|
7. Primary antibody. Incubate sections with the first primary antibody diluted in appropriate antibody diluent (buffer containing 5% NS) using an appropriate concentration and length of incubation.
|
|
8. Wash for 5 minutes in buffer.
|
|
9. Secondary antibody. Incubate sections for 30 minutes with biotinylated secondary antibody (5-10 µg/ml diluted in buffer containing 5% NS).
|
|
10. Wash slides for 5 minutes in buffer.
|
|
11. Avidin conjugate. Incubate sections with Fluorescein Avidin DCS (15-20 µg/ml diluted in buffer) for 5-10 minutes.
|
|
12. Wash slides for 5 minutes in buffer
|
|
Staining for Second Antigen
|
|
13. Avidin/biotin blocking step. Apply an Avidin/Biotin block according to Step 4. (This step must be done to prevent the interaction of the second set of labeling reagents with the first set of labeling regents).
|
|
14. Protein blocking step. Incubate sections for 20 minutes with 5% NS.
|
|
15. Blot excess serum from sections.
|
|
16. Primary antibody. Incubate sections with second primary antibody diluted in appropriate antibody diluent (buffer containing 5% NS) using an appropriate concentration and length of incubation.
|
|
17. Wash slides for 5 minutes in buffer.
|
|
18. Secondary antibody. Incubate sections for 30 minutes with biotinylated secondary antibody (5-10 µg/ml diluted in buffer containing 5% NS).
|
|
19. Wash slides for 5 minutes in buffer.
|
|
20. Avidin conjugate. Incubate sections with Texas Red® Avidin DCS (15-20 µg/ml diluted in buffer) for 5-10 minutes.
|
|
21. Wash slides for 5 minutes in buffer.
|
|
22. Mount with the appropriate VECTASHIELD® mounting media.
|
|
23. Observe under a fluorescence microscope.
|
|
NOTES:
|
|
1. Aldehyde-fixed tissues (e.g. formalin) tend to be autofluorescent and may make interpretation of specific fluorescein signal difficult.
|
|