Hi
My experiment is to look at the intracellular location of a small peptide. I have difficulties when I try to do the IF staining for the small peptide that has been uptaken by cells, and the main problem is the loss of signal after permeabilization.
The peptide is around 6k Da and labeled with Alexa flour 647. It was added to the cell culture media for 10min, then, cells were washed and fixed with 4% PFA, then permeabilized.
I have compared the signals of the live cell, 4% PFA fix-only cells, and fix+permeabilized cells, they all were treated with same peptide the signal loss happened mainly after the permeabilization process, and especially at the cell peripheral part. In the fix and permeabilized cells, the signal of the peptide is only shown in the central part of the cells.
My first thought is, maybe due to low MW, the fixation is not strong or long enough for the peptide, so I have tried different PFA concentration(1%-4%) and time (5min-2h), but increasing PFA concentration and time did not help.
I also tried 0.1%, 0.5%, 1% TX100 and saponin for the permeabilization, it seems saponin is better than TX100, but still have the issue of loss of peptide signal.
I am just wondering if anyone knows the optimized or specific fixation and permeabilization protocol for this kind of application with small peptides. Thanks
I understand the challenges you're facing with immunofluorescence staining of small peptides. Loss of signal after permeabilization could be due to inadequate fixation or issues with the permeabilization process itself. Here are some suggestions to optimize the protocol for staining small peptides:
- Consider trying alternative fixation methods such as cold methanol (-20 °C) or a mixture of methanol and acetone (1:1) for 10-15 minutes. Cold methanol or methanol:acetone mixture fixation can be more suitable for preserving small peptides and may improve retention of the labeled peptide in cells.
- Triton X-100 is a relatively harsh detergent that might remove the small peptide from cells during permeabilization. Since you mentioned saponin seems to work better, try using a lower concentration of saponin (e.g., 0.05% or 0.1%) and optimize the incubation time to minimize signal loss. You can also try other mild detergents, such as digitonin, at various concentrations (e.g., 0.01% to 0.1%).
- Perform a blocking step using a serum-free blocking buffer, such as 1% BSA in PBS, for 30 minutes to 1 hour before permeabilization to minimize non-specific binding and loss of signal.
- Optionally, consider co-incubation of the permeabilizing agent (e.g., saponin or digitonin) with the blocking buffer to reduce peptide loss during permeabilization.
- Ensure that the imaging settings are optimized to detect the Alexa Fluor 647 signal, as the signal intensity may be reduced after permeabilization. Use the same settings for acquiring images of live cells, fixed-only cells, and fixed + permeabilized cells for accurate comparison.
- Perform control experiments with an appropriate negative control (e.g., cells not treated with the labeled peptide) to ensure that the observed signal is specific to the peptide and not due to artifacts.
- If possible, consider using a larger peptide or a peptide with modifications that can improve its stability and retention in cells during fixation and permeabilization. This may require additional experimentation.
By adjusting the fixation, permeabilization, and blocking conditions, you should be able to optimize the protocol for detecting the small peptide in cells. Keep in mind that optimization might require multiple iterations, and it's important to be consistent with the conditions for each set of experiments for accurate comparison.
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