Depending upon the size of the sections, use appropriate containers, and volumes for incubation and washes. Antibody incubations need be in small volumes to save precious antibodies and wash volumes have to be generous to ensure proper wash of reagents. Use either a 12-well or 24-well plate for antibody incubations. For a 1 inch square section, use at least 1ml of solution in a 12-well dish; or use at least 0.75ml in a 24 well dish. Use a 6-well plate for blocking and washes at 6ml/well.
If sections are free-floating and have been stored in a cryopreservative solution, remove needed sections from solution into a large volume of PBS at room temperature. Wash for at least 10 minutes, shaking gently. Transfer sections to a large volume of fresh PBS and repeat wash before proceeding.
1. Block sections for 1 hour at RT, shaking, in appropriate Blocking Solution.
2. Transfer sections to wells containing primary antibody(s) and shake at 4° overnight. Dilute antibodies in Carrier solution.
3. Wash sections in Carrier solution for 5min, 15min, then 5min. at RT, shaking.
Note: Use generous wash volumes (eg. 6ml in 6 well plates) and make sure the sections are fully immersed and they are fully unfurled. It is necessary to wash away reagents entirely.
4. Incubate sections in secondary antibody(s) (1:100 works well) for 1 hr. at RT, shaking. Dilute secondaries in Carrier solution.
5. Wash sections in Carrier solution for 5min, 15min, then 5min. at RT, shaking. (Same precautions apply as in step 3 ).
6. Transfer one section at a time to a dish of PBS and mount section onto a gelatin-coated slide. Use a brush to remove wrinkles.
7. Dry back of slide and set slide on a 45°C warmer until completely dry (approximately 5 minutes).
8. Place 120µl of MOWIOL (Kept under vacuum overnight) on one side of the section in a thin line.
9. Hold coverslip flat on top of the mounting medium and gently touch the medium.
10. Lower slowly and let capillary action fill space.
11. Avoid air bubbles at all costs.
12. Store flat and dark overnight. Store long term in refrigerator in the dark.
