To begin, isolate the left atrium and pulmonary veins or PVs from the mouse's heart. Place the isolated tissue into a cryomold with the heart base and lung apex facing up. Untwist and untangle the PVs in case they are convoluted.
Then, fill the cryomold with the OCT compound. Using fine forceps, apply gentle compression on the PVs without moving them to remove residual air. Freeze the tissue in the OCT compound on dry ice.
For immunostaining, thaw the frozen mouse PV slides on a staining system for 20 minutes. Then administer several drops of a 4%paraformaldehyde-fixing solution onto the sections to secure the tissue to the slide. After incubation, move the slides to a vertical rack and submerge them in a container filled with PBS.
Set the container on a slow shaker for five minutes to rinse the slides. Using a liquid blocker pen containing xylol, encircle each section on every slide. And reorganize the slides on the staining system.
Using a Pasteur pipette, apply one or two drops of permeabilization solution onto each sample. Then, clean the Triton X-100 solution by washing the slides for five minutes in PBS. After rearranging the slides on the staining system, add one or two drops of the blocking buffer to each section, ensuring complete coverage.
Next, prepare one milliliter of the primary antibody mix consisting of the primary antibodies and blocking buffer. Discard the leftover blocking buffer from the slide. Administer two or three drops of the primary antibody mixture to each section.
After incubation, place the slides in a vertical rack and rinse the slides for five minutes in wash buffer while maintaining gentle agitation. Then, prepare one milliliter of the secondary antibody mix consisting of the secondary antibodies and washing buffer. Reposition the slides on the staining system and use a pipette to dispense two or three drops of the secondary antibody blend onto each section.
Subsequently, clean the slides three times for five minutes each in wash buffer while shaking. Next, add two or three drops of the Hoechst 33342 solution to each section arranged on the staining system. Place the slides in a vertical rack and rinse them for five minutes in wash buffer with gentle agitation.
Add one or two drops of fluorescent mounting medium to each slide and seal the slides with cover slips. The pulmonary vein orifice region at the left atrium and pulmonary vein junction, extrapulmonary veins, and interpulmonary veins were observed under 10X magnification. Specific cTnT signals with a typical muscular striation in the pulmonary vein orifice, extrapulmonary veins, and intrapulmonary veins, were observed at 40X magnification.
Cx43 was found in all three pulmonary regions and was primarily projected between neighboring cardiomyocytes. Cx43-related signals were observed on the polar side and the lateral side of the cardiomyocytes in the myocardial sleeves.
