Immunofluorescence Staining of Gel-Embedded Patient-Derived Diseased Fibrovascular Tissue

Take rehydrated fibrin gel-embedded, fixed, and permeabilized patient-derived diseased fibrovascular tissue. This tissue contains irregular neovascular structures with endothelial cells and pericytes.

Treat the tissue with a blocking solution to prevent non-specific antibody binding.

Incubate with primary antibodies targeting surface proteins on endothelial cells and pericytes.

Rinse with buffer to remove unbound antibodies, then incubate in buffer.

Following buffer wash, incubate with fluorescent dye-conjugated secondary antibodies to bind to the primary antibodies.

Wash with buffer to remove excess antibodies and incubate in buffer.

Following buffer wash, add a nuclear dye to stain cell nuclei. Rinse with buffer and deionized water to remove unbound dye.

Transfer the tissue onto a microscope slide.

Apply mounting media and seal with a coverslip containing quick-hardening mounting media.

After air-drying, store the slide at a low temperature. Then, image the tissue under an epifluorescence microscope to visualize the spatial arrangement of endothelial cells and pericytes within the neovascular structures.

Tilt the plate on a support and incubate the droplets in 500 microliters of blocking solution for two hours at room temperature. Prepare the primary antibody mixture according to the text protocol.

Then use a round-edged spatula to transfer the droplets to a round bottom 96-well plate. Incubate the droplets in 30 microliters of primary antibody mixture per droplet overnight at 4 degrees Celsius. The next day, transfer the droplets to a 12-well plate containing 2 milliliters of washing solution per well. Rinse the droplets with three 5-minute washes. Then wash the droplets with nine 30-minute washes.

The following day, rinse the droplets three times with PBS. Prepare the appropriate fluorophore conjugated secondary antibody mixture according to the text protocol. Transfer the droplets to a round bottom 96-well plate as previously demonstrated. And incubate the plate with 30 microliters of secondary antibody mixture for four hours at room temperature, protected from light.

After 4 hours, transfer the droplets to a 12-well plate containing 2 milliliters of washing solution per well. First, rinse the droplets with three 5-minute washes. Then wash the droplets with four 30-minute washes.

The next day, rinse the droplets five times with washing solution for 30 minutes and three times with PBS for 30 minutes. The next day, transfer the droplets to a round bottom 96-well plate as previously demonstrated. Counterstain the droplets with Hoechst nuclear stain for 30 minutes at room temperature. Transfer the droplets to a 12-well plate containing 2 milliliters of PBS per well. And rinse three times with PBS.

Next, apply a narrow layer of quick hardening mounting medium along the edges of a square cover glass. And allow it to dry for one minute. Then rinse a droplet by dipping it into a well of a 12-well plate containing deionized water. And transfer the droplet to a microscope slide.

Next, dispense 15 microliters of non-hardening anti-fade mounting medium onto the droplet. Gently position the cover glass over the droplet with the mounting medium facing the slide. And let it settle.

Let the slides dry for two hours at room temperature and store them at 4 degrees Celsius overnight. Finally, image the slides with an upright epifluorescence microscope equipped with an optical sectioning function at 20x or 40x objective.