Acute and Subacute Effects of Injury on the Canine Alveolar Septum: Methods (3)
The fixed lungs were washed with fresh 0.1-M phosphate buffer. Biopsy specimens from representative areas of the treated lungs were taken from areas marked by the India ink to insure that the area had been exposed to either papain or physiologic saline. Specimens were prepared for light microscopy and for scanning and transmission electron microscopy. Pieces of tissue for scanning electron microscopy, approximately 10 X 4 X 2 mm, were dehydrated in alcohol, critical point-dried in liquid CO.,, mounted on stubs, coated with carlx>n and gold-palladium and viewed in a Philips 305 electron microscope at 10 to 20 kV.
The tissue for transmission electron microscopy was carefully diced, postfixed in 1 percent osmium tetroxide in 0.1-M sodium cacodvlate for 1 h on a shaker and dehydrated in ethanol; ten pieces of tissue per lobe were embedded in Epon 812 by standard procedures. Sections ljim in thickness were cut from each block, stained with methylene blue and examined by light microscopy to identify tissue which contained India ink particles. From these blocks, thin sections (600 to 800 A, estimated with the use of a continuous interference color and thickness scale, in accordance with common practice), were examined at 75 kV by an RCA EMU 4B transmission electron microscope equipped with a low-magnifi-cation projector pole-piece. buy antibiotics online
An appropriate area from each specimen was selected for serial sectioning. The method of serial sectioning and montaging the whole circumference of alveoli previously has been described. In brief, an area that by light microscopy appeared to be representative was selected from each of the specimens.