Last updated: 06/06/19

For FISH protocol see: FISH with #1.5, 40 mm coverslip

1. Three washes with PBT for 10 minutes each.
2. Wash with 3% BSA-PBT 1 hour [1.5g BSA in 50ml PBT; 1% is equal to 1 g in 100 mL].
3. Add 35 µl primary Ab in 1% BSA-PBT to Bioptechs coverslip and cover with 22X30 coverslip, seal with rubber cement (if overnight).
   Hybridization cocktail:
   • 7 µL of 100 µg/mL Rabbit-LaminB1 (ab16048).
   • 7 µL of 50 µg/mL mouse-CyclinB1 (Invitrogen MA5-14319 is @ 200 µg/mL).
   • 21 µL 1% BSA in PBT
4. Incubate overnight 4C or at RT for 2 hours.
5. Three washes PBT 10min each
6. Add 35 µl secondary Ab in 1% BSA-PBT to Bioptechs coverslip and cover with 22X30 coverslip, seal with rubber cement (if overnight).
   Hybridization cocktail:
   • 7 µL of 1:40 Goat anti-rabbit IgG (H+L), CF568 (Biotium 20801).
   • 7 µL of 1:40 (50 µg/mL) goat anti-mouse, Alexa488 (Invitrogen A-11029, 2 mg/mL).
   • 21 µL 1% BSA in PBT
7. Incubate overnight 4C or at RT for 2 hours.
8. Two washes PBT 10 min each.
9. One wash in 1X PBS.