High-throughput Imaging and Analysis Workflow for Evaluating Skin Cell Phenotypes and Proliferation States in Tissue Samples

This protocol combines the multiplex optical imaging method, IBEX and Click-iT EDU labeling to detect and categorize dividing cell types in highly dynamic processes, such as skin repair. Moreover, we provide a novel open-source imaging processing pipeline for high throughput image acquisition and analysis. Coat the 24 well plate with 200 microliters of chrome aluminum gelatin and incubate the plate for 15 minutes on a rocker shaker.

Then completely remove the gelatin and dry the coated wells for 60 minutes in a 40 degrees oven. Cut the OCT-embedded tissue at 15 to 30 micrometer thickness at the cryostat and transfer it quickly into a coated well containing two milliliters of PBS. Carefully remove the PBS using a one milliliter pipette aiming to keep the tissue section centered in the well.

To do this effectively, slowly aspirate the PBS from the edges of the well, adjusting the pipette position as needed to prevent the tissue from shifting. Dry the tissue sections for one hour in a 37 degrees Celsius oven. Rehydrate with one milliliter PBS for five minutes.

Permeabilize the tissue with 500 microliters of 0.5%Triton for 30 minutes at room temperature. Wash the tissue briefly twice with PBS and perform the Click-iT reaction for 30 minutes at room temperature. Keep the samples protected from light.

Wash the tissue briefly twice with PBS. Block for one hour with a blocking buffer at room temperature. Add the primary antibodies for the first cycle.

And incubate the plate overnight at four degrees Celsius. Wash with PBS three times. Perform the secondary antibody staining.

For the first cycle, apply the secondary antibodies for the uncoupled primary antibodies together with a nuclear counterstain. Incubate the samples protected from light for 60 minutes at room temperature. Wash the tissue three times with one milliliter PBS for five minutes at room temperature.

Leave about 500 microliters of PBS in each well and bring the plate to the microscope. In the Meta Express software, click on Screening and then Acquisition Setup. Click on the Eject button.

Clean the bottom of the plate with 70%ethanol and insert plate into the microscope. Click the Load Plate button. Within the Objective and Camera tab, select the desired magnification.

As acquisition mode, select the confocal 51 micrometer slit mode. Within the Plate tab, select the plate model. Go to the Plates Sites Visit tab.

Under Site Options, click on fixed number of sites. Set the number of both columns and rows such that roughly the entire well is covered. Click on the overlap sites 10%Select only the first well for acquisition by right clicking the corresponding well on the plate map.

Go to the Acquisition Autofocus tab. For well-to-well autofocus, select the plate and well bottom option. For site autofocus, select the All Sites option.

Go to the Wavelengths tab and choose the number of dyes to be imaged in the current cycle. Go to the first wavelength tab. Choose laser with Z offset for the Z-positioning, and click the Calculate Offset button to set the correct imaging height.

Choose the image where the sample is best in focus. Press the Focus button. An in-focus image of the tissue is shown.

Set the illumination power to 50%Set the target max intensity to 2000 and press Auto Expose. For Z-series, choose Z-series and 2D projection image, and for shading correction, choose FL shading only. Repeat the steps for other wavelengths, but instead of laser with Z-offset, choose Z-offset from W1 and choose zero.

Go to the Z-series step and enter the desired step size. Click the Focus button. Drag the current position arrow until the bottom of the tissue is reached and click the Set B button.

Drag the position arrow to the top of the tissue and click Set T button. Click on F2, Stop, press Start Live again. Ensure that the stage is at the first well by left clicking on it.

Find the border of the tissue and mark all sites containing tissue for acquisition by right clicking. Go to the Run tab and type the name of the plate. Click on Save Protocol.

Set up the acquisition region for all the other wells. Run Control, Journal, start recording and then immediately Control, Journal, stop recording. Save the created journal and open it again via Control, Journal, Edit Journal.

In the left panel, go to Plate Acquisition, load protocol from file and click on Plate Acquisition to add those steps to the journal. Double click on the Edit Plate Acquisition, load protocol from file, and choose the protocol saved for the first well. Repeat the steps for each well to be acquired, making sure to always load the respective protocol.

Click on Run Journal to start the acquisition. 30 minutes before the end of the journal, start to prepare the bleaching reagent. Dissolve 10 milligrams of lithium borohydride in 10 milliliter of double distilled H2O and pass through a 0.22 micrometers filter.

Leave for 10 minutes. The solution should start to form bubbles. To ensure efficient bleaching, use the bleaching reagent within four hours after preparation.

Add the bleaching solution to the samples and incubate for 15 minutes whilst keeping the samples exposed to ambient lighting. Small bubbles should appear on the tissue. Wash the bleached samples three times with one milliliter PBS for five minutes, each at room temperature.

Add the primary antibodies for the next cycle and incubate overnight at four degrees Celsius. Multiplexed IBEX imaging combined with Click-iT EDU labeling reveals different structures and proliferating cell types in the wounded skin. EDU labeling on cryopreserved samples shows to be similar when performing antibody immunofluorescence labeling for KI-67 on paraffin-embedded tissue sections, as well as labeling cells with KI 67 using flow cytometry.

Using this protocol allows accurate detection of cell proliferation in complex biological processes. Moreover, the open source imaging processing pipeline does not require any programming experience and generates files that can be processed with non-commercial software, such as Fiji and quPath. Furthermore, this protocol does not require any expensive equipment and therefore, can be performed in most research settings.