Preparing Mouse Lung Tissue Sections for Isolectin B4 Staining to Evaluate Pulmonary Microvascular Density

To begin, place the mouse lung tissue in 4%paraformaldehyde, which is 10 times the volume of the tissue. After three hours of fixation, separate the five pulmonary lobes and embed the cranial, middle, and accessory lobes of the right lung separately, orienting in the direction of the vertical lobe-to-bronchus connection. Then cut the caudal lobe of the right lung into two pieces at four millimeters from left to right in the direction perpendicular to the lung-lobe-bronchus connection, and embed the slices in a wax block, with the cut surface facing upward.

To make three cuts in the left lung lobe, cut transversely above the main bronchial junction, two millimeters below the top. Make the second cut transversely above the main bronchial junction, five millimeters below the top. Make the third cut at the end of the lobe, eight millimeters below the top.

After discarding the uppermost piece of tissue, encase the remaining three pieces in a wax block, with the cut surface facing upward. Following refixation, rinse the tissue block under flowing water for 15 minutes. Sequentially immerse the tissue in the escalating ethanol concentrations, followed by xylene and soft wax.

Fill a mold with an appropriate amount of liquid paraffin and, using forceps, position the tissue in the embedding frame at the center. Place the mold and tissue in the freezing area of the embedding machine. When the paraffin starts to whiten slightly, quickly position the uncovered embedding frame on top of the mold and perform the second wax injection, sealing the holes at the bottom of the embedding frame.

Then transfer the entire mold to the freezing table. After securing the wax block onto the slicer, trim the block to reveal a smooth and flat cut surface. Rotate the microtome's wheel crank to cut slices with a thickness of four micrometers.

Smoothly place the removed sections onto the water surface sequentially. Once the sections are fully extended, separate the broken pieces at the junctions between each section. Tilt a glass slide underneath the section, and, upon contact, slowly and evenly lift the slide out of the water surface in a vertical motion.