Begin by embedding lung tissue and agarose and sectioning it using a vibratome. To the sliced tissues, add 300 microliters of the blocking agent. Incubate the plate at four degrees Celsius on a shaker with gentle agitation for 45 minutes.
Next, pipette 300 microliters of the PBS diluted primary antibody into each well. Incubate the plate overnight at four degrees Celsius with gentle shaking. Now gently pipette out the primary antibodies without touching the slice.
Wash the slices three times with 400 microliters of PBS. Next, pipette 300 microliters of the dilute secondary antibodies into each well. Remove the antibody solution after cold incubation for 90 minutes.
Then add the DAPI and tomato lectin 488 to the wells. Incubate the mixture for 30 minutes. After removing DAPI and tomato lectin, wash the slices in PBS for three cycles of 10 minutes.
With a brush, transfer the slices to a slide. Place three drops of mounting medium on the slide and mount a cover slip over it before imaging. The lung sections of mouse, pig, and human samples were immunofluorescently stained for SMA, elastin, and LEA.
The SMA and elastin were observed to be distributed across structures such as alveolar ducts, blood vessels, bronchioles, and alveoli. Comparison of type II pneumocytes distribution in adult and infant humans was performed. The type II alveolar cell antibody HT2-280 had a strong affinity to the pneumocytes cell surface.
The infant sample yielded a significantly higher type II pneumocytes count. However, the infant sample also exhibited a significantly higher scaffold coverage than the adult. The number of type II pneumocytes based on scaffold coverage was also significantly higher in the infants.
