Dear researchers
We are preparing for superresolution microscopy. I read some papers where the cells are prepared with the use of a special cytoskeletal buffer (CB) and followed by NaBH4 incubation. I have two questions.
1 What is the purpose of the CB [10 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.1, 150 mM NaCl, 5 mM ethylene glycol tetraacetic acid, 5 mM MgCl2, 5 mM glucose]
2 Why is the NaBH4 incubation?
Thanks for an answer.
I haven't the foggiest. But my view is that any washes or treatments prior to fixation could already induce some changes to the cytoskeletal structure. I would therefore start out with the simplest of protocols, i.e. just take off the medium, and add the fixative (e.g. 4% PFA and 1% sucrose in 0.1M phosphate buffer pH 7.4, preheat to avoid temperature changes during fixation).
What kind of super-resolution imaging are you setting up?
Hi Dr. Specht, Thanks for your answer. We are interested in desmosomal proteins. In the group of Mattheyses (Stahley, JCS 2016) a thorough investigation of desmosomal proteins was published. A different desmosomal protein has gotten our attention. We are preparing to study this in superresolution microscopy. The conditions they use are quite rough. A superresolution microscopic investigation on hemidesmosomes was published in 2015 from the group of Jalink (Nahidiazar in JCS). This group has used a different buffer for fixation and preparation: cytoskeletal buffer is what they call it. This buffer seems to be more gentle. No I was interested why this buffer contains extra magnesium, while the EGTA must scavenge the Ca++. It is mildly buffered with MES instead of phosphates, why is that? What is the purpose of sucrose in this buffer? Osmolarity probably, but maybe there is a different reason. Duco Kramer
Dear Duco,
if you are studying a crowded environment like desmosomes this makes more sense. Is the protein that you are interested in extracellular? If so, the preparation of hemidesmosomes may be necessary to increase the labeling efficiency. Will you be using antibodies for labeling and STORM super-resolution imaging ?
Best of luck,
Chris
Dear Chris,
The POI is intracellular, so we have to permeabilise the cells prior to labeling. I think of using saponin for this, but a little triton may also be a possibility. We will indeed go for dSTORM-imaging. We do this with a group specilialized in dSTORM. They provided us with a set of 2nd Ab suitable for this.
Sounds good. In that case, you just need to optimise your immunolabeling. If you get nice labeling with conventional imaging, the super-resolved images will also look great. Best of luck,
Chris
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