Hematoxylin and eosin was used for general morphology. Mowry and
Marand Alcian blue/periodic acid–Schiff (PAS), Gomori aldehyde
fuchsin, Fullmer and Lillie oxidation aldehyde fuchsin, and Verhoeff
techniques were used for demonstrating different classes of
glycosaminoglycans and elastic fibers at different stages of maturity.
McGee–Russel alizarin red S technique and the Pizzolato silver method
were used to demonstrate calcium. Congo red staining was used to
exclude amyloid. Oil red O was used on frozen sections to demonstrate
lipid.
The distribution of TIMP-3 (Triple Point Biologics, Forest Grove, OR),
type IV collagen (Dako Ltd., High Wycombe, England), type V collagen
(Chemicon Ltd, Harrow, UK), type VI collagen (Chemicon Ltd.),
fibrillin (Chemicon Ltd.), fibronectin (Dako Ltd.), laminin (Dako
Ltd.), and elastin (Elastin Company, MO) were investigated
using a standard biotin–streptavidin biotin, alkaline phosphatase
complex method (Dako Ltd.). The alkaline phosphatase label was
visualized as a red final reaction product (Vector Ltd., Peterborough,
England). Nuclei were weakly stained with Mayer’s hematoxylin. TIMP-3,
elastin, and fibronectin antibodies had been raised in rabbit, and the
others were mouse monoclonals.
Antigen retrieval was utilized in all cases. Sections for TIMP-3 and
fibrillin required pressure cooking. Sections for fibronectin, laminin,
and elastin demonstration were exposed to 0.1% trypsin for 15 minutes
at 37°C, whereas types IV, V, and VI collagen sections received 0.4%
pepsin treatment for 60 minutes at 37°C. Electron microscopic
semithin sections were stained with toluidine blue, and ultrathin
sections were stained using lead citrate and uranyl acetate.
Special stains, immunohistochemistry, and electron microscopy also were
carried out in a normal donor eye from a 72-year-old normal female
donor for comparison. This eye was fixed in 10% formol saline and was
treated exactly as the SFD eye as described above. The son of the SFD
donor, aged 57 years, also was affected; his right fundal picture
showed widespread, drusenlike structures at the posterior pole (
Fig. 1A ), whereas his left fundal picture showed a disciform macular scar
(Fig. 1B) .