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An En Bloc Staining Protocol Improves The Preservation of Lamellar Bodies in Alveolar Type II Epithelial Cells for Transgenic Mice Expressing Modified Pulmonary Surfactant Protein B

Published online by Cambridge University Press:  02 July 2020

C.-L. Na ,
D. C. Beck ,
J. S. Breslin ,
S. E. Wert  and
T. E. Weaver
C.-L. Na
Affiliation:
The Children's Hospital Research Foundation, Division of Pulmonary Biology, Department of Pediatrics, 3333 Burnet Avenue, Cincinnati, OH45229
D. C. Beck
Affiliation:
The Children's Hospital Research Foundation, Division of Pulmonary Biology, Department of Pediatrics, 3333 Burnet Avenue, Cincinnati, OH45229
J. S. Breslin
Affiliation:
The Children's Hospital Research Foundation, Division of Pulmonary Biology, Department of Pediatrics, 3333 Burnet Avenue, Cincinnati, OH45229
S. E. Wert
Affiliation:
The Children's Hospital Research Foundation, Division of Pulmonary Biology, Department of Pediatrics, 3333 Burnet Avenue, Cincinnati, OH45229
T. E. Weaver
Affiliation:
The Children's Hospital Research Foundation, Division of Pulmonary Biology, Department of Pediatrics, 3333 Burnet Avenue, Cincinnati, OH45229
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Extract

The extraction of lipids and phospholipids during dehydration and plastic embedding steps results in poor preservation of the phospholipid rich lamellar bodies (LB) in alveolar type II epithelial cells. To achieve better retention of phospholipids, we combined inflation fixation and an en bloc staining protocol using 4% aqueous uranyl acetate (UA), thereby improving the preservation of the LBs for both the wild type and transgenic mice expressing modified pulmonary surfactant protein B (SP-B; Akinbi et al., 1997).

Lungs of 6-8 week-old mice were inflation fixed (Bunkingham and Weyder, 1981) with ice cold 2% paraformaldehye and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (SCB), pH 7.3, postfixed in fresh fixative at 4 °C overnight, incubated with 1% osmium tetroxide in 0.1 M SCB at room temperature for 2 hours, and stained en bloc with 4% aqueous UA overnight.

Type
Specimen Preparation
Information
Microscopy and Microanalysis , Volume 4 , Issue S2: Proceedings: Microscopy & Microanalysis '98, Microscopy Society of America 56th Annual Meeting, Microbeam Analysis Society 32nd Annual Meeting, Atlanta, Georgia July 12-16, 1998 , July 1998 , pp. 852 - 853
Copyright
Copyright © Microscopy Society of America

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References

1.Akinbi, H. T. et al., J Biol Chem, 272 (1997) 9640.CrossRefGoogle Scholar
2.Bukingham, K. W. and Wyder, W. E., Toxiclogic Path, V9 (1981) 17.CrossRefGoogle Scholar