3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

The scope of this research is to study the morphology and spatial distribution of parasites in the retinal vasculature using advanced imaging techniques. In order to provide a detailed view to demonstrate the morphological characteristics of parasites, the study described a new approach combining the retroorbital injection of fluorescent agent, immunofluorescent staining, and tissue clearing treatment to advance research in our field.

Compared to the pre-clearing state of the mouse retinal tissue, the background signals of blood vessels and parasites labeled with fluorescent tomato lactin and PDGFR-beta are significantly reduced after tissue clearing, facilitate a more precise observation of their spatial relationship.

Our results indicate that these techniques are highly compatible for demonstrating the parasites in retinal vasculature in the whole mount retina. The combination of these histological techniques is an effective approach to visualize the morphological characteristics of parasites in detail from a three-dimensional view.

[Instructor] To begin, place the anesthetized mouse in the right lateral recumbency with its head facing to the left. Gently press two fingers on the periorbital area to expose the mouse's left eye. Using a one-milliliter syringe equipped with a 27-gauge needle, gently pierce about two to three millimeters into the orbital venous sinus, ensuring the bevel of the needle is facing forward at a 45-degree angle. Inject 0.1 milliliters of fluorescent tomato lactin into the orbital venous sinus. After five minutes of retroorbital injection, euthanize the mouse and use surgical scissors to open the thoracic cavity. Insert a 24-gauge needle two millimeters into the left cardiac ventricle through the left ventricular apex. Perform perfusion at a rate of three milliliters per minute with 20 milliliters of 0.9% physiological saline, followed by 20 milliliters of 4% physiological saline. Inject 0.1 milliliters of 0.9% formaldehyde in 0.1 molar phosphate buffer. Next, place the sacrificed mouse on its side and remove the skin covering the eyes with scissors. Enucleate the eyes with scissors and forceps. Cut the optic nerve and surrounding tissues and lift out the eye. Transfer the eye to a 12-well plate for fixing in 4% formaldehyde in 0.1 molar phosphate buffer for two hours. After fixation, cryoprotect the eye in 25% sucrose in 0.1 molar phosphate buffer at four degrees Celsius until it sinks to the bottom of the solution. Using a plastic pastor pipette, transfer the eye into a Petri dish containing 0.1 molar phosphate buffer. Pierce the edge of the cornea with sharp scissors, cut around the cornea and iris, and discard them. Using forceps, remove the lens and vitreous humor. Then, use fine forceps to pull the cup-shaped retina away from the center of the eye. Rinse the retina with 0.1 molar phosphate buffer in a clean Petri dish. After isolating the cup-shaped mouse retina, incubate it in a 20-hour incubator with a 2% Triton X-100 solution in 0.1 molar phosphate buffer overnight at four degrees Celsius. The next day, transfer the retina into the blocking solution and rotate at 72 RPM on the shaker overnight at four degrees Celsius. Then, place the retina into a microcentrifuge tube containing the primary antibodies of goat anti-PDGFR beta in dilution buffer. Rotate the tube on the shaker for two days at four degrees Celsius. After two days, wash the retina twice with washing buffer at room temperature. Place the retina in the washing buffer and transfer it to the shaker overnight at four degrees Celsius. The next day, transfer the retina into a microcentrifuge tube containing a solution of one milligram per milliliter secondary antibody of donkey anti-goat 488 and 0.2 nanograms per milliliter fluorescent nuclear stain DAPI. Rotate the tube on the shaker at 72 RPM for five hours at four degrees Celsius. Wash the retina in a six-well plate with washing buffer for one hour twice at room temperature. Incubate the retina in washing buffer on the shaker overnight at four degrees Celsius. The next day, transfer the retina to the tissue clearing reagent and gently rotate it on the shaker at 60 RPM for one hour at 37 degrees Celsius. After the tissue clearing, rinse the retina with 0.1 molar phosphate buffer in a clean Petri dish. Using spring scissors, make four radial incisions reaching approximately 2/3 of the radius of the retina to create a petal shape. Flatten the retina and mount it on a microscope slide with the inside of the retina facing up. Use a small piece of absorbent paper to remove any excess phosphate buffer. Circle the mounted retina with a spacer. Fill the gap with fresh tissue clearing reagent and place a cover slip on top. Scan the montage views of the labeled retina using the panoramic tissue slice scanner. Take the higher magnification images using an imaging system equipped with a 2X and a 10X lens with numerical apertures of 0.40 and 0.95, respectively. Capture 50Z stack images from the labeled retina in two micrometer frames. Sequentially click Set Start Focal Plane, Set End Focal Plane, Set Step Size, choose Depth Pattern, Image Capture, and Z-Series to integrate all images into a single in-focus image using projection and topography mode. Next, capture images using the green fluorescence at 499 and 519 nanometers, red fluorescence at 591 and 618 nanometers, and blue fluorescence at 401 and 421 nanometers. Sequentially select Add New Surfaces, choose Volume Settings, choose Source Channel, adjust display, adjust surface angle, and snapshot to import the Z-Stack confocal images into analysis software, and reconstruct the images in the three-dimensional pattern. The tissue transparency of the whole mount retina was increased after the tissue clearing treatment compared to the retina without tissue clearing. Retinal vasculature labeled with tomato lectin and retinal parasites labeled with PDGFR-β were visible both before and after tissue clearing, but the background noise was reduced following tissue clearing. Retinal parasites were observed along the vascular tree, showing a gradational distribution from the central to peripheral regions of the retina. The three-dimensional Z-Stack images from the cleared retina showed parasites tightly wrapped around retinal vessels of varying diameters with stronger PDGFR-β staining in capillaries.