09/26/23 Immuno procedure

Immunofluorescence Procedure Tu 9/26/23

Materials

• PBS-Tw-Az for rinsing
• Little beakers
• forceps
• enough for 4 humid chambers
  • petri dish
  • filter paper
  • parafilm
• fixed coverslip of HTC116 cells, 1 per student
• Mixed primary antibodies
  • Mouse anti-Lamin A
  • Rat anti-alpha tubulin
• Mixed secondary antibodies
  • Goat anti mouse 488 (green)
  • Goat anti rat TRITC (red)

To mix primaries:

• Lamin A:
  • Use 4 aliquots of 5 µL each
  • Add 125 µL PBS-Tw-Az-BSA to get (slightly less than) 2X strength
  • total volume: 520 µL
• Alpha Tubulin:
  • Use 3 aliquots of 4 µL each
  • Add 200 µL PBS-Tw-Az-BSA to get 2X strength
  • total volume = 600 µL
• Mix 1:1, and aliquot ~104 µL per pair. Make 10 aliquots

To mix secondaries

• Green anti-mouse:
  • 2 1µL aliquot
  • 300 µL PBS-Tw-Az-BSA
  • total volume: 602 µL
• Red anti-rat:
  • 2 5 µL aliquots
  • 260 µL PBS-Tw-Az-BSA each
  • total volume: 530 µL
• Mix 1:1, and aliquot ~106 µL per pair. Make 10 aliquots