Multiplexed Immunofluorescence Assay for Spatial Assessment of Senescence Markers in vivo

The spatial analysis of senescent cells is important for understanding their contribution to processes such as aging, cancer, immunity, and normal tissue homeostasis. Detection of senescent cells relies on profiling multiple biomarkers. Multiplex immunofluorescence imaging allows the high throughput profiling of these within a single tissue section.

To begin, verify that the 5-micrometer thick formalin-fixed, paraffin-embedded, or FFPE, tissue sections with a maximum size of 4 x 2 centimeters square are centrally mounted on positively-charged microscopy slides and are clearly visible within the coverslipless slide holder's viewing window. Perform the sample preparation steps, either manually using a decloaking chamber or automatically using an automated stainer. Place the prepared slides into the coverslipless slide holder.

Align the top and right edges against the metal stops. Lock the slide in place using both hands at the same time to turn the plastic cams to the closed padlock position. Verify the slide is seated completely flat and does not rock within the holder.

Place the ClickWell, insert chamber over the slide, and pinch the sidebars with two hands to limit pressure and prevent breakages. Load the click well with 2 milliliters of mounting media containing 50%glycerol in phosphate-buffered saline via the inlet port. Add a barcode label to the left-hand side of the slide in either orientation, then proceed for imaging.

For region selection and autofluorescence analysis, open the Mx Workflow version 4204, multiplex imaging acquisition software. Click on Tools to select Protocol Publisher. In the Multiplex Protocol Publisher pop-up, enter the protocol name.

Click on Create Setup Round, followed by add Next Protocol Round to create multiple rounds of biomarker labeling. Click Publish, then select OK in the pop-up window. Acquire the Cy3 channel for virtual H&E images.

Click on Slide, then Create New Batch. After selecting the protocol name, enter the slide ID.Click on Add Slide and then select Create Batch for creating a multiplex experiment workflow. Open the Cell DIVE acquisition software.

Click on Load to load the slide into the microscope. Under the Tools dropdown menu, click on the grid icon to define the desired regions and use the Move tool to reposition the defined regions. Next, use the Draw tool to deselect any fields not required, ensuring that each field of view has at least two neighboring fields of view to prevent stitching issues, and click Save Regions.

In the Mx Workflow window, click on Viewer QC to open the Cell DIVE viewer page and visualize the tissue image in 10x magnification. Click on the image correction icon to adjust the image brightness and click Submit QC to select Pass and Reload Slide in the pop-up window. Once the image reloads, click on Add ROI to draw and adjust the regions of interest.

Select the desired fields of view and repeat the addition of ROI. Next, click Send to Lab. Load the slide to start the 20x autofluorescence image acquisition.

Under the Imaging QC menu, select the checkbox corresponding to the specific Slide ID, then click Viewer QC.Zoom into the loaded image to scan for out-of-focus regions or artifacts. Check the background images for each channel, which will be subtracted from the final biomarker signal. Click Submit QC, then select Pass and Exit to generate background images.

Prior to sequential biomarker staining, centrifuge the antibodies at 13, 000 G for one minute at room temperature to limit artifacts. Prepare the antibody solution at a 1 in 200 dilution in phosphate buffered saline, or PBS, containing 0.3%bovine serum albumin, or BSA. Remove the mounting media by pipetting out from the ClickWell port.

And wash thrice with 2 milliliters of PBS containing 0.01%Tween 20 or PBST for 5 minutes. Add 350 microliters of antibody solution into the port to ensure full sample coverage within the staining chamber. Incubate for one hour at room temperature.

Next, pipette out the antibody solution from the ClickWell port. And wash thrice with 2 milliliters of PBST for 5 minutes. Add 2 milliliters of mounting media.

Insert the ClickWell holder into the microscope and load the slide to start 20x biomarker imaging. Perform the imaging QC using the blend editor and image correction options and submit the image QC evaluation. Pipette out the mounting media from the ClickWell port and wash thrice with 2 milliliters of PBST for 5 minutes.

Prepare the dye inactivation solution freshly before use. After removing PBST, add 2 milliliters of dye inactivation solution for each slide. Incubate for 15 minutes in the slide holder.

Pipette out the dye inactivation solution from the ClickWell port and wash with 2 milliliters of PBST for 5 minutes. Add 2 milliliters of mounting media and insert the ClickWell holder into the microscope to initiate automatic round-specific 20x autofluorescence imaging. Progress the workflow by passing QC steps as directed on screen upon completion.

Repeat the sequential staining, dye inactivation and multiplex imaging for as many markers as required, and continue until completion. Use HALO software to select various channels for visualizing the localization of markers at different regions of the tissue core. Click Analyze and select Entire Image to analyze the quantitative metrics for individual markers.

Click on Results to view the total number of cells in the analyzed tissue core and the subset of cells expressing specific markers. Compared to the automated slide stainer-based method, the manual decloaking chamber-based method resulted in a higher frequency of tissue artifacts characterized specifically by tissue detachment as well as lost or slipped cores. Quantification performed with digital pathology software on representative pancreas tissue revealed that p16 positive cells were associated with lower LB1 levels and vice versa, demonstrating a profile consistent with senescence associated marker patterns.

A Z-score normalized heat map illustrates the distribution of the p16 positive lamin-B1 negative senescence phenotype across various tissues, revealing high concentrations of senescent cells in the lung, adrenal and pituitary glands corresponding with elevated p16 and diminished LB1 levels. Conversely, the appendix, lymph node and spleen exhibited low senescence, maintaining high LB1 and low p16 expression. This protocol allows the probing of a combination of senescence, structural and immune biomarkers within a single microscopy slide.

By combining multiple markers within a single sample, spatial analysis of tissue architecture and senescent cell distribution can be performed. Future application include the large scale profiling of senescence biomarkers across multiple tissue types and integration with spatial omics to better resolve cellular heterogeneity.