Detecting both mRNA and protein in the same sample using a combination of Hybridization Chain Reaction (HCR) and Immunofluorescence (IF) is a powerful technique, but it requires careful optimization to ensure that both signals are preserved and can be distinguished. Here are some recommendations for setting up your protocol:

Fixation:

Paraformaldehyde (PFA): PFA is commonly used for fixation as it preserves both cellular structure and protein antigens well. It is suitable for both HCR and IF. Fixation with PFA is typically done at 4% for 10-30 minutes at room temperature or overnight at 4°C.

Methanol (MetOH): Methanol can also be used for fixation and is particularly good for permeabilizing cells, which is important for mRNA detection. However, it can sometimes cause protein denaturation. If using MetOH, it’s often followed by a rehydration step in PBS.

Permeabilization:

For HCR, you’ll need to permeabilize the cells to allow the probe to access the mRNA. This can be done with a higher concentration of detergent like 0.5-1% Triton X-100 in PBS after fixation.

Ensure that the permeabilization step is gentle enough not to disrupt the protein epitopes.

Blocking Solution:

The concentration of blocking solution can vary, but a common starting point is 5-10% normal serum (from the species in which your secondary antibodies were raised) or 2-5% BSA in PBS or TBST.

The blocking step is crucial to reduce non-specific binding of antibodies.

Stripping:

If you are concerned about antibody cross-reactivity or need to reuse the same sample for multiple primary antibodies, you might consider a stripping step. This can be done using gentle methods such as glycine buffer or more aggressive methods like using a commercial antibody stripping buffer.

However, stripping can potentially remove or damage the mRNA signal, so it’s often better to plan your experiments to avoid the need for stripping.

Protocol Tips:

Sequential Detection: Start with HCR to detect mRNA, then proceed with IF for protein detection. This order is typically chosen because the HCR reaction conditions are less harsh and less likely to affect the subsequent IF steps.

Probe Design: Make sure your HCR probes are designed to minimize cross-reactivity with cellular proteins or other RNA species.

Antibody Validation: Use validated primary and secondary antibodies for IF. Ensure that the fluorophores used for HCR and IF are compatible and can be distinguished by your imaging system.

Controls: Include appropriate positive and negative controls for both HCR and IF to validate your assay.

Imaging: Use a confocal microscope or a microscope equipped with deconvolution software to minimize bleed-through between channels and to accurately localize signals.

References:

Choi, H., et al. (2018). “Simultaneous Detection of mRNA and Protein in Single Cells with Hybridization Chain Reaction and Immunofluorescence.” Cell Chemical Biology, 25(7), 950-958.

Stavis, C., et al. (2018). “Simultaneous Multiplexed Detection of RNA and Proteins in Single Cells.” Cell Reports, 25(1), 63-71.

Remember that protocols can vary greatly depending on the specific cell type, antibodies, and probes used, so you may need to adjust these recommendations to fit your experimental setup. Always start with positive controls to optimize each step before applying it to your samples.