Smooth muscle cell markers in developing rat lung

JJ Mitchell, SE Reynolds, KO Leslie, RB Low… - Am J Respir Cell Mol …, 1990 - atsjournals.org
JJ Mitchell, SE Reynolds, KO Leslie, RB Low, J Woodcock-Mitchell
Am J Respir Cell Mol Biol, 1990atsjournals.org
Materials and Methods Guaranteed, timed-pregnant Fischer 344 rats were obtained from
Taconic Farms (Germantown, NY) at day 14 of gestation. The animals were housed in a
barrier facility and provided food and water ad libitum throughout the study. Rats were killed
and tissues prepared for study on days 15 through 21 of gestation (pseudoglandular and
canalicular stages); days 1, 3, 5, 8, 11, and 13 of postnatal life (primary and secondary
saccular stage); and during the alveolarization period on postnatal days 15, 19, 25, and 34 …
Materials and Methods
Guaranteed, timed-pregnant Fischer 344 rats were obtained from Taconic Farms (Germantown, NY) at day 14 of gestation. The animals were housed in a barrier facility and provided food and water ad libitum throughout the study. Rats were killed and tissues prepared for study on days 15 through 21 of gestation (pseudoglandular and canalicular stages); days 1, 3, 5, 8, 11, and 13 of postnatal life (primary and secondary saccular stage); and during the alveolarization period on postnatal days 15, 19, 25, and 34, as well as from adult animals. Pulmonary tissue from three animals was examined at each point. These specimens were obtained from at least two different litters to permit examination oflittermates at more than one time point and to control for developmental variation due to litter size. Animals were killed by sodium pentobarbital overdose. Uteri were removed from pregnant animals and transferred to a petri plate containing chilled phosphate-buffered saline (PBS) on the stage of a dissecting microscope. After rapid dissection, the heart/lung preparation or in some cases the entire thoracic region of the fetus was fixed en bath in 100% ethanol for immunocytochemical analyses. During the first 2 wk of postnatal life, the lungs were dissected free of other tissue, perfused with PBS by syringe via the heart, and gently inflated with fixative from a 3-cc syringe via the trachea before immersing in the fixative bath, Lungs from older animals were first perfused in situ with PBS via the right ventricle, then the trachea was cannulated and the tissue inflation fixed at a pressure head of approximately 20 cm H20. The cannula was subsequently tied off and the lungs were immersed in fixative.
Tissue was allowed to fix in ethanol for 48 h prior to embedding in paraffin. The entire sample from the fetal and postnatal days 1 and 3 was embedded. For samples taken later in development, the right lung or lower lobe of the right lung was used for the immunohistochemical analysis. The general histologic appearance of the tissue and direct assessment of the developmental stage was obtained from standard hematoxylin and eosin (H & E) stained companion sections. One to three slides per block were processed for immunostaining with each antibody or control serum. Four-micron sections were cut, deparaffinized in two changes of xylene, and rehydrated through 100%, 95%, 75%, and 50% ethanol into PBS. Endogenous peroxidase was inactivated by immersing the sections in a methanol-3% H20 2 solution (4: 1) for 30 min at room temperature. Blocking steps included incubation with 3% bovine serum albumin for 30 min at 37 C and in 10% normal goat serum for 30 min at 37 C. The a-smooth muscle actin-specific monoclonal antibody, anti-asm-1 (8), in hybridoma cell medium supernate, was diluted 1: 50 in PBS, applied to the sections, and. reacted overnight at 4 C. The polyclonal guinea pig antivimentin (9) and antidesmin (9) sera were used at 1: 100 dilution, while
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