This method is helpful to detect, and co-localize multiple biomarkers in a single cryosection including markers that are not normally detectable in paraffin sections. Using this experimental staining method, the staining time is significantly reduced and the method can be performed in any laboratory. Demonstrating this method will be Dinesh Jaishankar.
He is a skilled postdoc from the lab, and also the Immunotherapy Assessment Core, Facilities Manager. When selecting antibodies for the analysis, consider the excitation and emission filter sets available on the semi-automated imaging system. And after testing various combinations of fluorophore conjugated primary antibodies, prepare fluorophores to be used so that you have minimal spectral overlap as suggested in the table.
For multiplex staining, prepare a cocktail of antibodies with compatible fluorophores at predetermined optimal delusions, and add the cocktail to the tissue sections of interest. For single marker staining, add the primary conjugated antibody only to the slide. For all stainings, include a control unstained slide that undergoes the same staining procedure without the addition of a primary conjugated antibody.
After a one-hour incubation at room temperature protected from light, wash the slides two times with PBS for five minutes per wash. After the second wash, counter stain the multiplex stained slides with DAPI for seven minutes at room temperature protected from light, followed by two five-minute washes in PBS. To create a spectral library, set the lamp power of the multispectral imaging system to 100%and open the microscope operating software.
Select Edit Protocol and new protocol. Enter a protocol name and select Fluorescence under imaging mode. Then provide a study name, and load the single stained slides onto the stage.
Click Edit Exposure, and use the auto-focus and auto-expose options to adjust the exposure times and acquire snapshots for the single stained slides. After imaging the single stained slides, save the protocol. And in the machine learning software under the Build Library's tab, load each single stained image.
Select the appropriate floor and click Extract. The software will automatically extract the fluorescent signal selected in the floor. To save the extracted color, click Save to store.
A new group may be created or the extracted color can be stored to an existing group. For multispectral imaging, once the spectral library has been created and verified, adjust the focus and exposure times on the multiplex stained slide as demonstrated, and opens scanned slides to create a new task. Provide a slide ID and select a protocol.
Under task, select whole slide scan to perform a whole slide scan on the multiplex stained slide. In the whole slide scan image, select regions of interest across the image. These regions will be scanned using the previously set exposure times on the multiplex slide to be used for spectral unmixing and analysis.
In the microscope operating software, select acquire MSI fields under task, and then click Process Slide to acquire regions of interest at a 20x magnification. Once the regions of interest have been acquired, in the machine learning software, click File and Open under the manual analysis tab to load the multiplex stained images. Select the spectral library source, and click Select Floors to choose the previously saved spectral library.
Click File and Open to load the unstained image. Then click the autofluorescence ink marker icon and draw a line or region on the unstained slide to identify autofluorescence within the tissue. Then under the Edit Markers and Colors tab, assign names for each marker and click Prepare All.
After verifying the spectrally unmixed image, in the machine learning software, select the cytoplasm membrane and marker options from the panel. And using the ellipsis button, select use this signal to find, to configure the marker to detect either the nuclei, cytoplasm or membrane. when a cytoplasmic or membrane marker is chosen, select the Use the Signal to assist in nuclear splitting option.
The software will automatically detect and create individual masks for the nucleus, cytoplasm and membrane. Use the pathology view for a specific marker and the configuration options in the software to adjust the masks, and click Segment All to create a cell segmentation map. After cell segmentation, select the markers to phenotype the positive cells.
Additionally, select at least five cells that are not stained with the chosen marker to phenotype the negative cells. Then click Train Classifier to train the software to automatically detect all of the cells stained with the selected markers within the image. A phenotype map will be created.
As these images demonstrate, antibodies validated for flow cytometry could be used to visualize tissue staining using the liquid crystal tunable filter in the semi-automated imaging system. Here a spectrally unmixed image of frozen human tonsil can be observed that includes labeling of B-cells and Proliferating cells within the follicular germinal center, and the inter follicular T-cell region. The pathology views option allows visualization of the individual staining pattern for each marker within the tissue of interest, suggesting that the multiplex staining method and spectral imaging work on frozen tissue.
As demonstrated on a frozen mouse tumor section, multispectral imaging can also be used to visualize tumor infiltrating T-cells, and other myeloid cell lineages including tumor associated macrophages. Cell segmentation and phenotype maps, and the number of stained cells for each marker analyzed by the software can be generated, demonstrating that the software can be used for quantification of the multispectral staining on frozen tissues. For someone new 6to the technique, it's important to get to know the specific features of your fluorochromes, and prepare quality library slides for spectral unmixing with minimal spectral overlap.
It's also important to perform antibody titration. In our opinion, this method can provide bedside information about the efficacy of immunotherapy for cancer and other therapeutic applications, particularly when timing is of the essence.
