Biology Reference
In-Depth Information
8. Immediately add 12-30 μL of primary (1°) antibody (e.g., rabbit anti-LPS,
diluted 1:200-1:1,000 in PBS) directly onto the coverslip ( see Note 12 ). The
antibody solution must stay on the coverslip. Leave at room temperature for
15-30 min depending on antibody.
9. Rinse twice with PBS.
10. Wash three times for 10 min each with PBS.
11. Remove PBS thoroughly by aspiration and make sure coverslip is not touching
sides of well.
12. Immediately add 12-30 μL of secondary (2°) antibody, for example,
AlexaFluor568-conjugated goat anti-rabbit antibody diluted in PBS ( see
Note 13 ). The antibody solution must stay on the coverslip. Leave at room
temperature for 15-30 min depending on antibody.
13. Rinse twice with PBS.
14. Wash three times for 10 min each with PBS.
15. Permeabilize cells and block by incubating with SS-PBS, 250-400 μL/well, for
15-20 min at room temperature.
16. Remove SS-PBS thoroughly by aspiration and make sure coverslip is not
touching sides of well.
17. Immediately add 12 μL of primary (1°) antibody (e.g., mouse anti-LPS, diluted
1:200-1:1000 in SS-PBS) ( see Note 14 ). The antibody solution must stay on the
coverslip. Leave at room temperature for 20-60 min depending on antibody.
18. Rinse once with PBS.
19. Wash three times with PBS, 10 min each.
20. Incubate briefly with SS-PBS for 2-5 min.
21. Immediately add 12 μL of appropriate secondary (2°) antibody, for example,
if AlexaFlur568 conjugated antibodies were used in step #12, then use
AlexaFluor488-conjugated goat anti-rabbit antibody diluted in PBS ( see
Note 13 ). The antibody solution must stay on the coverslip. Leave at room
temperature for 20-60 min depending on antibody.
22. Rinse twice with PBS.
23. Wash three times for 10 min each with PBS. It is crucial to remove all unbound
antibody before proceeding to the next step.
24. Optional: Rinse once with distilled H 2 O.
25. Mount coverslips, cells down, on clean glass slides using 5-10 μL Mowiol ( see
Note 6 ) per coverslip.
26. Allow Mowiol™ to harden overnight at room temperature in the dark.
27. Observe cells using a fluorescence microscope equipped with the appropriate
filters/laser lines for the fluorophores used.
4. Notes
1. Glycerol stock cultures should be kept at -80 °C. Bacteria for experiments should
be picked from colonies grown on LB plates not more than 1 week old and
stored at 4 °C. The SL1344 strain is streptomycin resistant, so can be grown on
LB plates containing 100 μg/mL streptomycin.
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