Post-fixation Following Antibody Labeling
A post-fixation step can be valuable. Herein, you fix cells a second time after staining in order to improve the stability
of the label. This can prevent the label from detaching and floating the imaging medium.
Imaging Buffer dSTORM
(please see: G.T. Dempsey, Nature Meth 2011 and S. van Linde, Nature Meth 2011)It is recommended to freshly
prepare the imaging buffer every day. The oxygen scavenger system will only last for a few hours and is mainly needed
for Cyanine dyes like Alexa 647 and Cy5 (Rhodamines and Oxazines do not require it.)
Imaging Buffer
100µl PBS 10x (Phosphate buffered saline ; e.g. from Sigma: D1408)
100µl MEA (Cysteamine Hydrochloride) stock concentration 1M (e.g. from Sigma M6500-25G) (toxic!!)
Optional:
Oxygen scavenger system (for cyanine dyes)
500µl Glucose 20% (e.g. in solution from Sigma (49163-100ML))
25µl Glucose Oxidase (e.g. 24 mg/ml GluOx from Aspergillus niger; Sigma G0543-50KU)
5µl Catalase (e.g.: 12.6 mg/ml Catalase from Bovine liver; Sigma C3155-50 MG)
Add H2O to final volume of 1ml
Very important: adjust pH to 7.5-8.5 with 5 M NaOH or 4.5 M KOH
(To test pH you can use pH paper in the indicated range)
Note
1 M MEA (Sigma M6500-25G): 0,113g in 1ml
Store MEA (solid) at 4ºC. Prepare fresh as 1 M working stock solution in water. This stock can be kept at 4ºC and
used within 1-2 weeks of preparation. Alternatively you can freeze small aliquots at -20ºC and keep them for several
months.
The MEA concentration depends highly on the fluorophore and the experimental condition. Therefore the best
concentration has to be tested by trial and error for each sample (between 10mM and 100mM).
If you use Cysteamine (not the Hydrochloride) you have to use 37% HCl to adjust the pH to 7.5-8.5
As a general advice:
• If blinking rates are too high: increase MEA concentration and/or increase pH
• If blinking rates are too low: decrease MEA concentration and/or decrease pH
White Paper
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