09/26/23 Immuno procedure
09/26/23 Immuno procedure kdorfman Thu, 09/21/2023 - 21:19Immunofluorescence 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
- 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