Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

This protocol is particularly useful in large animal models of lung transplantation and regeneration, such as pigs, where we can sample small pieces over time. This technique can help us better understand the spatial biology at play in these models. This technique produces minimally-processed lung slices suitable for immunofluorescence imaging without the need for antigen retrieval, and has been effective with all antibodies tested to date.

This procedure can be adapted to cut slightly thicker sections, say around one millimeter, which can be then used in light sheet fluorescence microscopy to study the spatial protein expression in a more three-dimensional volume. Our research is investigating factors influencing lung transplant rejection, and developing therapies to reduce rejection using our pig model. This technique helps understand spatial protein changes affecting transplant outcomes post-surgery.

We're currently using this technique to explore the spatial protein expression landscape in our pig lung transplant model. We hope that it will help us to better understand the mechanisms behind our experimental therapies. The key to succeeding here lies in vibratome sightings, high blade amplitudes, and lower force speeds are key things to look at if the sectioning goes badly.

To begin, fix the porcine lung tissue in 4%paraformaldehyde at room temperature for 48 hours. Then transfer the lung tissue to a 50 milliliter conical tube containing 30 milliliters of PBS with 0.01%sodium azide. Keep a plastic cup, fine forceps, and sterile scissors ready.

Heat the agarose in a microwave until it reaches boiling point. Then cool it down to 42 degrees Celsius in a water bath. Store it in the water bath until ready for use.

Remove the lung biopsy from the PBS-azide solution. Use fine forceps and sterile scissors to carefully dissect the lung lobes into smaller blocks. Next, cut down the middle of a plastic cup.

Pour a small amount of agarose at the bottom of the cup. Place the lid of a 50 milliliter conical tube with its rim removed on the agarose surface. Store this set up at four degrees Celsius for five minutes to allow the agarose to solidify.

Once the agarose has solidified, remove the plastic cup from the refrigerator. Now pour about one milliliter of liquid agarose onto the lid. Then gently place the lung tissue block on the lid.

Once the agarose has semi solidified, slowly submerge the lung tissue in liquid agarose. Refrigerate the setup at four degrees Celsius for about 15 minutes until the agarose becomes solid. Use a sharp blade to carefully remove any excess agarose surrounding the lung tissue.

To begin, embed the lung tissue in agarose. To prepare the lung tissue for sectioning, first, attach each tissue block to the specimen disc of the vibratome with super glue and add ice to the surrounding bath. Then carefully install the specimen disc into the buffer tray and fill the tray with PBS.

Next, set the vibratome parameters, keeping the thickness at 200 micrometers, the frequency at 100 hertz, the amplitude of the blade at 1.3 millimeters, and the forward speed of the blade between 0.02 and 0.03 millimeters per second. Commence the sectioning of the lung tissue. Once enough slices have been created, use a brush to gently lift the tissue slice from the vibratome tray.

Transfer the slices to a 24 well plate with PBS-azide solution. Preserve the lung slices at four degrees Celsius. Begin by embedding lung tissue and agarose and sectioning it using a vibratome.

To the sliced tissues, add 300 microliters of the blocking agent. Incubate the plate at four degrees Celsius on a shaker with gentle agitation for 45 minutes. Next, pipette 300 microliters of the PBS diluted primary antibody into each well.

Incubate the plate overnight at four degrees Celsius with gentle shaking. Now gently pipette out the primary antibodies without touching the slice. Wash the slices three times with 400 microliters of PBS.

Next pipette 300 microliters of the dilute secondary antibodies into each well. Remove the antibodies solution after cold incubation for 90 minutes. Then add the DAPI and tomato lectin-488 to the wells.

Incubate the mixture for 30 minutes. After removing DAPI and tomato lectin, wash the slices in PBS for three cycles of 10 minutes. With a brush, transfer the slices to a slide.

Place three drops of mounting medium on the slide and mount a cover slip over it before imaging. The lung sections of mouse, pig and human samples were immunofluorescently stained for SMA, elastin and LEA. The SMA and elastin were observed to be distributed across structures such as alveolar ducts, blood vessels, bronchials, and alveoli.

Comparison of type two pneumocyte distribution in adult and infant humans was performed. The type two alveolar cell antibody HT2-280 had a strong affinity to the pneumocyte's cell surface. The infant sample yielded a significantly higher type two pneumocyte count.

However, the infant sample also exhibited a significantly higher scaffold coverage than the adult. The number of type two pneumocytes based on scaffold coverage was also significantly higher in the infants.