All procedures used in these studies followed the tenets of the
Declaration of Helsinki and the ARVO Statement for the Use of Animals
in Ophthalmic and Vision Research. Informed consent was obtained before
obtaining human tissue samples. Full ethical approval was granted for
all portions of this study by the Lothian Health Research Ethics
subcommittee and by the Western General Hospital NHS Trust Research and
Development Ethics Committee. Rat eyes were obtained from healthy adult
male Lister-Hooded rats and paraffin-embedded. Human eyes (obtained
from Glaucoma Research Foundation, San Francisco, CA, and the Queen
Victoria Hospital, East Grinstead West Sussex, UK) were cut in lateral
and medial parasagittal planes, and sections were paraffin-embedded.
Horizontal sections (5-μm-thick) were cut using a microtome (Leitz
GmBH, Wetzlar, Germany) and sections placed on
3-aminopropyltriethoxysilane (APES 2%; Sigma, St. Louis, MO)–coated
slides. Sections were deparaffinized by immersion in histoclear (2 × 10 mins; Fisher Scientific, Loughborough, Leicestershire, UK).
Histoclear was removed by washing in ethanol (100% × 2 minutes;
Merck, Poole, UK). Sections were rehydrated by immersion in graded
alcohols (100%, 100%, 95%, 85%, 70%, 50%, 30% ethanol). Ethanol
was removed by washing in sodium chloride (0.9%). This was followed by
immersion in Triton-X (0.3%; Koch Light, Suffolk, UK) in 1×
phosphate-buffered saline (1× PBS for 15 minutes) after which sections
were washed twice in 1× PBS (5 minutes). Tissue sections were then
digested in trizma–HCl (100 mM, pH 8; Sigma), EDTA (50 mM; Sigma)
containing proteinase K (30 minutes, 37°C; Sigma), then washed in
glycine (0.1%; Merck) in 1× PBS. Sections were then postfixed in
paraformaldehyde (4%; Fisher Scientific), washed in 1× PBS (2 ×
5 minutes) followed by acetylation in acetic anhydride (0.25%; Sigma)
in triethanolamine (0.1 M, pH 8; Sigma), washed in 1× PBS (1 × 3
minutes), dehydrated in graded alcohols, and air-dried. Sections were
incubated with prehybridization buffer made up of diethylpyrocarbonate
water, sodium chloride (5 M), trizma base (1 M), 50× Denhardt’s
(Sigma), salmon testes DNA (Sigma), EDTA (250 mM; Sigma), and yeast
tRNA (GIBCO–BRL Products, Paisley, UK) in deionized formamide
(50°C × 2 hours; Sigma). Hybridization was then carried out by
incubation with 35S-labeled riboprobe (1 ×
106 cpm) in hybridization buffer containing
diethylpyrocarbonate water, sodium chloride (5 M), trizma base (1 M),
50× Denhardt’s, salmon testes DNA, EDTA (250 mM), and yeast tRNA in
deionized formamide (50°C × 16 hours). After hybridization,
sections were washed in SSC (15 minutes) and incubated with RNase A
(100 μg/ml, 37°C for 1 hour; Sigma). Sections were then washed to
increasing stringencies to a maximum of 0.1× SSC (60°C for 1 hour).
After dehydration through graded alcohols, sections were placed against
hyperfilm βmax (2 weeks at 4°C; Amersham) and
autoradiographs developed. After this, sections were dipped in
photographic emulsion (NTB-2; Kodak, Rochester, NY) and exposed
(4°C for 3 weeks) before being developed and counterstained with
hematoxylin and eosin (Sigma).
Areas of specific mRNA expression on tissue sections were
identified by the appearance of silver grains. Grain counting (SEE-Scan
Image Analysis Systems UK) was performed on antisense and corresponding
sense sections. Background counts (counts from sense sections) were
subtracted from antisense counts and results expressed as multiples of
background. Positive control sections were used as follows: rat
hippocampus (GR, MR and 11β-HSD type 1), rat kidney (11β-HSD type
2), human cerebellum (GR), human liver (11β-HSD type 1), and human
kidney (MR and 11β-HSD type 2).