Based on the EMBL Advanced Course Super-resolution microscopy – July 2014
(PFA/ Formaldehyde) fixation followed 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
a. 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).
2. Fix with 2% PFA in PBS for 15 min.
a. 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.
3. Rinse 3 x with PBS
4. Wash 3x with PBS for 5 min
a. Additional washing steps enhance results in super resolution imaging.
5. Permeabelise with 0.1% Triton in PBS for 10 min.
a. 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.
6. Rinse 3 x with PBS
7. Block with 2% BSA in PBS for 1h
a. 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 the antibodies as the serum helps to preserve the structure.
8. Incubate with primary antibody for 1h
a. Higher antibody concentration and longer incubation rimes might increase
background staining. One alternative is overnight incubation at 4°C.
9. Wash with 3 x PBS for 5 min
C. Walther
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Based on the EMBL Advanced Course Super-resolution microscopy – July 2014
10.
11.
12.
13.
a. This is the absolute minimum time for the washing step, previous rinsing steps might
help but the time shouldn’t be shortened.
Incubate with your secondary antibody for 1h
a. 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.
Wash 3 x with PBs for 5 min.
Store your samples in PBS at 4°C
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. Walther
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