Question: Is the Fixed RNA Profiling assay compatible with intracellular staining?
Answer: We have not tested or validated intracellular staining in the Fixed RNA Profiling Solution. The detection of intracellular antigens requires reagent and protocol modifications. Additional optimization of staining and wash buffers is required for optimal assay performance. When a 10x Demonstrated Protocol becomes available for intracellular staining, it will be tested for compatibility.
Although intracellular staining is not officially supported by 10x Genomics, limited additional guidance can be provided to help enable these experiments. These recommendations are likely to be changed when an official 10x Demonstrated Protocol becomes available. We advise performing a small pilot study before committing to large-scale experiments. Re-optimization may be required for additional antibodies.
The fixation recommendations provided in the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling Demonstrated Protocol may need to be modified. Instead of a 1 hr fixation with 4% Formaldehyde at room temperature, we have found during limited testing in-house that a shorter fixation time prior to antibody labeling may be helpful. Samples should be fixed with 4% Formaldehyde for 10-20 minutes following the guidance provided in the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling, washed with staining buffer, stained with antibodies of interest, and then fixed again for 1 hr with 4% Formaldehyde following the guidance provided in the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling. The second fixation may be necessary for keeping the antibody attached during probe hybridization and for optimal assay performance.
Labeling for cell surface proteins should be performed prior to fixation and staining of intracellular proteins. The presentation of the cell surface antigens are often affected by fixation. Some antibodies recognizing cell surface markers may not bind to fixed antigens.
Antibody labeling and washing should be performed with PBS + 1% BSA. We recommend using nuclease-free BSA for optimal assay performance. Samples should be washed two times, by adding 1.5 ml chilled PBS + 1% BSA to the labeled cells. Centrifugation speed and time depends upon the sample type.
Alternatively, other intracellular fixation kits with permeabilization buffers that may be compatible. These include BioLegend’s Cyto-Fast™ Fix/Perm Buffer Set (cat# 426803) or BioLegend’s Fixation buffer (cat# 420801) paired with Intracellular Staining Permeabilization Wash Buffer (cat# 421002). Samples should be fixed again for 1 hr with 4% Formaldehyde following the guidance provided in the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling . Other fixation buffers are untested and unsupported. The permeabilization in the 10x Demonstrated protocol may be appropriate for cytoplasmic targets, but for nuclear targets a nuclear antibody staining kit with more intense detergents may produce better results.
This guidance is meant as a starting point. Fixation times should be optimized and may vary depending on the antibodies used.
Please note: These workflow modifications are untested and not supported on the Fixed RNA Profiling Assay.
Products: Fixed RNA Profiling Gene Expression