Dual-modality Molecular Cartography: Integrating Multiplex mRNA Detection with Protein Imaging Mass Cytometry

We focus on special biology to further understand the molecular mechanisms of ovarian cancer in the hopes to find new therapeutical targets and biomarkers to predict patient survival. One of the current IMC challenges includes dissection of lower abundance protein, the labor of antibody validation and titration, as well as the complex and time-consuming pixel classification during analysis. Begin by removing excess liquid from the prepared FFPE slides.

Take the humidity control tray out of the oven, and place the slide holder into the tray. Then, vortex RNA-ISH Amplifier 3 thoroughly, and add enough reagent to completely cover each tissue section on the slides. Close the tray, and insert it into the hybridization oven for 30 minutes at 40 degrees Celsius.

During incubation, prepare the metal oligonucleotide mix by diluting each oligo at a one-to-15 ratio. Vortex the metal oligo mix thoroughly, and leave it at room temperature until further use. Now, remove the humidity control tray from the oven.

Take the slide holder out of the tray, and place the tray back into the oven. Then, place the slides into a clear slide holder that has been pre-washed with nuclease-free water. Wash the slides in wash buffer under slight agitation for two minutes.

After removing excess liquid from the slides, take the humidity control tray out of the oven, and place the slide holder into the tray. Add the mixed metal oligonucleotides prepared earlier to fully cover each tissue section. Now, close the tray completely, and insert it into the hybridization oven for 45 minutes at 40 degrees Celsius.

Remove the humidity control tray from the hybridization oven, take out the slide holder, and return the empty tray to the oven. To block nonspecific binding sites, add a sufficient volume of blocking buffer to fully cover each tissue section on the slide, and incubate the slides at room temperature for 30 minutes. While the slides are incubating, prepare the antibody mix by diluting the antibodies using the antibody diluent buffer.

Once the incubation is over, discard the buffer from the tissue slides, and dispense enough of the previously prepared antibody mix to completely cover the tissue sections. Then, place the slides into a humidity chamber, and incubate them overnight at four degrees Celsius. To prepare a fresh Iridium intercalator solution, make a one-to-2, 000 dilution from a 500-micromolar stock solution using RNase-free TBS.

Place the slides into a clear slide holder that has been pre-washed with nuclease-free water, and wash the slides in TBS-T buffer using slight agitation for five minutes. Then, to stain the slides, add the prepared Iridium working solution, and incubate for five minutes at room temperature. After that, place the slides into a clear slide holder that has been pre-washed with nuclease-free water.

Wash the slides two times in TBS-T buffer with slight agitation for five minutes. Then, dip the slides quickly in double-distilled water to prevent salt crystallization from TBS on the tissue. Dry the slides under a chemical hood for 10 minutes, and store them at four degrees Celsius until image acquisition.

Load the prepared slide into the ablation chamber of the imaging mass cytometry system. Capture a panorama image of the slide to identify the region of interest. Using the image acquisition software, mark the desired regions of interest on the panorama image.

Apply the acquisition template in the image acquisition software. And adjust the laser power based on the sample type. Now, start the laser ablation process.

Measure the quantity of each isotope using the detector, which converts the data into digital form for analysis. The activation status of CD8 T cells was assessed using co-detection of granzyme B at both mRNA and protein levels, along with interferon-gamma positivity. A cancer-associated fibroblast was shown to express MFAP5 protein and POSTN mRNA, both markers of cancer-associated fibroblast aggressiveness.

A CD4 T cell was shown to co-express IL17A and IL6 mRNA, consistent with TH17 cell identity. CD4 T cells were observed expressing IL13 mRNA. Keratin-positive CD47-positive tumor cells were shown near a CD8 T cell.

An activated CD8 T cell was shown to express CD25 protein. Comparable mRNA staining of THBS2 and CD47 was observed between metal-conjugated and fluorescent-labeled protocols.