IF protocol following FISH

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:

    1. 7 µL of 100 µg/mL Rabbit-LaminB1 (ab16048).
    2. 7 µL of 50 µg/mL mouse-CyclinB1 (Invitrogen MA5-14319 is @ 200 µg/mL).
    3. 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:

    1. 7 µL of 1:40 Goat anti-rabbit IgG (H+L), CF568 (Biotium 20801).
    2. 7 µL of 1:40 (50 µg/mL) goat anti-mouse, Alexa488 (Invitrogen A-11029, 2 mg/mL).
    3. 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.

For imaging protocol see: Day-of-experiment protocol, including fluidics

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