Based on the EMBL Advanced Course Super-resolution microscopy – July 2014

(PFA/ Formaldehyde) fixationfollowed by antibody staining

Reagents:

-PBS, pH 7.4

- 2% Paraformaldehyde in PBS

- 0.1% Triton in PBS

- Bovine Serum Albumin (BSA)

Procedure:

All steps are performed at room temperature.

  1. Rinse 3x with PBS
  2. Cells should be washed, culture medium removed; tissue should be dissected and cleaned from parts hindering image acquisition. Use established protocols, if they are known to work for imaging. Treat samples gentle and work quick to avoid damaging the sample (cell death and decomposition).
  3. Fix with 2% PFA in PBS for 15 min.
  4. Fixation is a critical step, as it defines how well the strictures are preserved. With increasing resolution this steps becomes more critical. PFA is a common fixative but not always the best. Some research in literature and optimisation might be required. An alternative might be 5 min incubation in ice cold (-20°C) 100% methanol, which does not require additional permeabilsation steps. Hence step 5 and 6 can be ignored.
  5. Rinse 3 x with PBS
  6. Wash 3x with PBS for 5 min
  7. Additional washing steps enhance results in super resolution imaging.
  8. Permeabelise with 0.1% Triton in PBS for 10 min.
  9. Crucial step to reveal primary antibodies. Lower concentration/ shorter incubations may better preserve the structure but compromise labelling density. High labelling densities are more important in super-res. imaging as in diffraction limited imaging.
  10. Rinse 3 x with PBS
  11. Block with 2% BSA in PBS for 1h
  12. Blocking can be performed with different reagents and should prevent unspecific labelling of the antibody. It is also advisable to use blocking reagents while incubating with theantibodies as the serum helps to preserve the structure.
  13. Incubate with primary antibody for 1h
  14. Higher antibody concentration and longer incubation rimes might increase background staining. One alternative is overnight incubation at 4°C.
  15. Wash with 3 x PBS for 5 min
  16. This is the absolute minimum time for the washing step, previous rinsing steps might help but the time shouldn’t be shortened.
  17. Incubate with your secondary antibody for 1h
  18. You might need to adopt/ optimize the antibody concentration. A good starting point for Alexa Fluor 488, ATTO 488 is a 1:100 dilution of commercially available ones, otherwise 5x higher than the recommended dilution. This again can also be done overnight at 4°C.
  19. Wash 3 x with PBs for 5 min.
  20. Store your samples in PBS at 4°C
  21. Mount directly before imaging using specific mounting buffer (GLOX, which needs to be prepared fresh).

Adding fiducial markers:

  1. Sonicate the 5 ml stock solution of tetraspecks (0.1mm) for 15 min
  2. After cell fixation and before permeabilisation remove PBS solution from washing and add fresh PBS (1ml)
  3. Put chamber/ coverglass on a bench rocker at low speed
  4. Add sonicated tetraspecks direct to chamber
  5. Incubate 30 min
  6. Wash 3x with PBS and proceed with staining.

C. Waltherpage 1