Post-fixation, it is recommended to perform approximately 7 minutes of 0.1 M Glycine or 0.1% (w/v) Sodium Borohydride during fluorescence labeling in super-resolution microscopy to reduce background fluorescence.
How exactly do the properties of both these compounds help play the same role while preforming the actual experiment? Are there any good sites or papers I can refer to?
Sodium Borohydride treatment helps reduce the background particularly when preceding chemical fixation used Glutaraldehyde. The orginal paper on the subject is this:
Clancy B, Cauller LJ (1998) "Reduction of background autofluorescence in brain sections following immersion in sodium borohydride". J Neurosci Methods 83:97–102
We had found that Sodium Borohydride treatment helps reduce the background AND also seems to improve photoactivation behavior of mEos fluorescent protein in PALM experiments:
Kopeck BG et al, 2011 "Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes", PNAS, 109(16):6136-41.
and
Paez-Segala MG et al, 2015, "Fixation-resistant photoactivatable fluorescent proteins for CLEM", Nat Methods, 12(3):215-8.
The trick is that the Sodium Borohydride MUST be very fresh to be effective. Once mixed, the solution produces bubbles for about 10-15 minutes. It must be used during that time, after it stops bubbling, its tops being effective.
Sodium borohydride reduces the aldehydes that have not reacted with protein in the sample so that they are no longer reactive, preventing them from covalently crosslinking to antibodies to your sample. Glycine, as a primary amine, reacts with any free aldehydes much the way a protein would, neutralizing any remaining aldehyde groups.
Sulekha Nair Hi Sulekha, did Glycine work for you? It would be nice to hear your experience.
Hi Surya Prakash Rai
I regularly use 0.3M glycine along with BSA/NGS during the blocking step and it does a great job
Atefeh Mohammadi Hi Atefeh, thanks for your response. How long do u block for and does it also for super-resolution microscopy? By the way can i get the protocol for preparing 0.3M glycine (pH=7.3 in PBS or ?)?
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH