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.