Silver:Immunofluorescence

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Materials

• 37% Formaldehyde
• 1 M DTT
• DAPI stock solution (1 mg/ml, 1000x)
• VectaShield mounting medium
• Clear nail polish
• Slides and coverslips
• 0.1 M potassium phosphate buffer pH 6.5
  • Solution A: 5.44 g KH₂PO₄ into 200 ml ddH₂O
  • Solution B: 6.96 g K₂HPO₄ into 200 ml ddH₂O
  • For pH 6.5, combine 68.5 ml (A) with 31.5 ml (B), bring up to 200 ml with ddH₂O, test pH and adjust with KOH
• Solution P
  • 0.1 M potassium phosphate buffer pH 6.5, and 1.2 M sorbitol
  • Add 21.9 g of sorbitol to 85 ml potassium phosphate buffer, mix (heat a little) and then top off to 100 ml for 1.2 M sorbitol, filter to sterilize
• Antibody Blocking Buffer
  • 1 ml 1 M Tris pH 9.0 (0.1 M Tris pH 9.0)
  • 300 µl 5M NaCl (0.15 M NaCl)
  • 500 µl FBS (5% FBS)
  • 300 µl 10% Triton-X 100 (0.3% Triton X-100)
  • 7.9 ml ddH₂O
• Antibody Wash #1
  • 1 ml 1M Tris pH 9.0 (0.1 M Tris pH 9.0)
  • 300 µl 5M NaCl (0.15 M NaCl)
  • 8.7 ml ddH₂O
• Antibody Wash #2
  • 1 ml Tris pH 9.5 (0.1 M Tris pH 9.5)
  • 200 µl 5M NaCl (0.1 M NaCl)
  • 500 µl 1 M MgCl₂ (50 mM MgCl₂)
  • 8.3 ml ddH₂O

Protocol

1. Grow 5 ml yeast to early log phase (5 x 10⁶ – 1 x 10⁷ cells/ml)
2. Move 5 ml culture from glass tube to 15 ml conical. Add 700 μl 37% formaldehyde, seal the tube with parafilm, and incubate at 30°C for 1 h
3. Pellet cells, 2 min at 2,500 rpm, wash 2 x 5 ml 0.1 M phosphate buffer and 1 x 5 ml Solution P
4. Resuspend pellet in 1 ml Solution P, cells can be stored at 4 °C for a while
5. Take 500 μl of your cells, add DTT to 25 mM (12.5 µl) and incubate at 30°C for 10 min.
6. Pellet cells, 2 min at 2,500 rpm and resuspend with 500 μl Solution P
7. Add 20 μl zymolyase (10 mg/ml) and 10 μl lyticase (Resuspend 50,000 units lyticase (one bottle) in 1 ml ddH₂O) for every 500 µl cells. Incubate at 30°C on rotator, 10-20 min is a good time range.
8. While zymolyasing, coat slide wells with 0.1% polylysine. Incubate 10 min at RT. Rinse 2x with ddH₂O and air dry.
9. Pellet cells, 1 min, 2,500 rpm and resuspend gently in 500 μl of Solution P
10. Wash 2 x 500 μl of Solution P
11. Apply 20 μl cell suspension to each well, incubate 5 min RT
12. Gently aspirate off the excess cells and permeablize them with 20 µl 0.5% Triton-X 100 in Solution P for 5 min.
13. Aspirate off Triton and rinse 1 x 500 μl with Solution P
14. Block cells in antibody blocking buffer for 30 min to 1 h at RT
15. Add primary antibody diluted into antibody blocking buffer and incubate overnight at 4°C
16. Wash wells (RT):
    • Ab wash #1 for 10 min
    • Ab wash #1 for 30 min
    • Ab wash #2 for 10 min
    • Ab wash #2 for 30 min
17. Add secondary antibody diluted in antibody blocking buffer, 1 h RT
18. Repeat washes (from step 16)
19. Stain nuclei with DAPI solution for 5 min at RT in the dark (DAPI stock = 1 mg/ml, 1000x, dilute in PBS)
20. Wash cells 2 x 5 min in the dark with Antibody wash #2
21. Aspirate off Antibody wash #2 and let air dry in the dark for a few minutes
22. Apply VectaShield mounting medium (prewarmed to RT), apply coverslip, and seal with clear nail polish
23. Publish results in Cell