Robust visualization of phosphorylated RIPK3 and MLKL, two key signaling events during necroptosis induction, has been technically challenging. The presented tyramide amplification protocol enables sensitive detection of these two molecules. The signal amplification step lowers the detection threshold, allowing robust and reproducible detection of phosphorylated RIPK3 and MLKL.
Begin seeding the 90, 000 ZBP1-expressing HT-29 cells in the full strength McCoy's 5A medium in a one centimeter square surface area well plate, allowing high-end microscopy. Use an end volume of 200 microliters per well. Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide.
Once the cells reach 70 to 80%confluency, infect the cells in 200 microliters preheated, full-strength McCoy's 5A medium containing herpes simplex virus one, encoding a rim mutant ICP6 at a multiplicity of infection of five. Incubate the cells with the virus for nine hours to induce necroptosis. As a positive control for necroptosis induction, stimulate the cells for four hours with 200 microliters of preheated necroptosis-inducing cocktail.
As a negative control, add 22 microliters of 10 micromolar GSK840 preheated at 37 degrees Celsius to inhibit RIPK3 kinase activity. Include this inhibitor in an untreated condition and after necroptosis stimulation to prevent the autophosphorylation of serine 227. To fix the cells, remove the medium and wash the cells with 200 microliters of PBS.
Then remove the PBS and add 150 microliters of 4%paraformaldehyde equilibrated at room temperature. Incubate the cells at room temperature for 30 minutes. After fixation, remove the 4%paraformaldehyde, and wash the cells three times with 200 microliters of PBS.
The samples can be stored in excess of PBS at four degree Celsius overnight until further processing. Remove the PBS from the plate, and incubate the cells with 100 microliters of permeation buffer at room temperature for 30 minutes on a tilting laboratory shaker. After incubation, remove the permeabilization buffer, and wash the cells with 100 microliters of wash buffer.
Incubate the imaging chamber with wash buffer for five minutes at room temperature on a tilting laboratory shaker. To prevent the non-specific binding of primary antibodies, incubate the plate with 100 microliters of blocking medium at room temperature for two hours on a tilting laboratory shaker. Next, wash the wells three times with 100 microliters of wash buffer with five minutes of incubation at room temperature on a tilting laboratory shaker.
After removing the wash buffer, add 100 microliters of the primary antibodies to the imaging chamber. Ensure to include a no primary antibody condition to visualize the potential background of the tyramide signal amplification, and to set the masking threshold. Incubate the chamber overnight at four degree Celsius.
Remove the primary antibody mix, and wash the wells three times with 100 microliters of wash buffer with five minutes of incubation at room temperature on a tilting laboratory shaker. After removing the wash buffer, incubate the cells with 100 microliters of HRP-labeled secondary antibody, recognizing the species of the primary antibody to be amplified for 30 minutes on a tilting laboratory shaker. During antibody incubation, prepare the biotinylated tyramide mix.
To trigger the enzymatic activity of HRP, add an oxidizing substrate to the TSA buffer freshly before use. Then dilute the biotinylated tyramide in the prepared TSA buffer using the optimized dilution. After incubation with HRP-labeled secondary antibody, wash the wells three times with wash buffer with five minutes of incubation on a tilting laboratory shaker.
After removing the wash buffer from the well, add diluted biotin-tyramide to the well to an end volume of 100 microliters. Incubate the reaction at room temperature for 10 minutes on a tilting laboratory shaker, followed by three wash buffer washes. Prepare the staining mix containing the secondary antibody, coupled streptavidin, and nuclear stain and wash buffer.
Remove the wash buffer and incubate the plate with 100 microliters of staining mix at room temperature for one hour on a tilting laboratory shaker. Keep the imaging chambers shielded from light from this step onward. After staining, consecutively wash twice with wash buffer, and rinse the wells two times with PBS.
Store the samples in excess of PBS at four degree Celsius to preserve the staining until imaging. The imaging chamber is now ready to be visualized on a confocal microscope. The inclusion of tyramide signal amplification enabled the robust detection of RIPK3 phosphorylated at serine 227 in the cytosol of herpes simplex virus one encoding a rim mutant ICP6 infected cells.
A laser power of 2%was sufficient for sensitive detection. In standard indirect immunofluorescence, a higher laser power remained insufficient for the detection of phospho-RIPK3. In quantification of the three-dimensional Z-stack images, the infected cells showed an approximately 20-fold increase of voxels positive for phosphorylated RIPK3 over mock-treated cells.
Increased phospho-RIPK3 staining of wild-type herpes simplex virus one infected cells compared to mock-treated cells indicated that the rim domain of ICP6 is unable to block RIPK3 phosphorylation at serine 227 completely. GSK840 binds to the kinase domain of RIPK3, and prevents RIPK3 phosphorylation at serine 227 upon ZBP1 activation, confirming the specificity of the tyramide signal amplification-mediated phospho-RIPK3 detection method. The mock-treated cells showed low in slightly punctuated cytosolic staining of MLKL phosphorylation at serine 358, while strong staining was detected in the cytosol, nucleus, and plasma membrane of the infected cells.
The standard indirect immunofluorescence required 40%laser power for the specific detection of a phospho-MLKL signal in the infected cells. In contrast, tyramide signal amplification increased detection sensitivity of phospho-MLKL, thereby lowering the necessary laser power to 6%3D Z-stack image quantification showed an approximately 90-fold increase in the number of voxels positive for phospho-MLKL using tyramide signal amplification, compared to a seven-fold increase using the standard method, indicating improved detection threshold and sensitivity for phospho-MLKL. Similar to herpes simplex virus one, influenza A virus infection triggered ZBP1-dependent necroptosis.
Tyramide signal amplification allowed the robust detection of phospho-RIPK3 and phospho-MLKL, indicating necroptosis. To interpret the confocal staining, several staining controls need to be included. This entails a no primary condition, single stains, as well as a positive control.
By adapting this protocol, multiple targets can be amplified using sequential TSA. This would allow the detection of phosphorylated RIPK3 and MLKL within the same cell. Sensitive detection of necroptosis biomarkers can be insightful for ongoing ZBP1 research on autoinflammation or virus infection in which the executed ZBP1-dependent cell death pathways still need to be confirmed as either necroptosis, apoptosis, or pyroptosis.
