TGF-β2 concentrations were determined in aqueous humor obtained
from 24 rd mice (2, 3, 7, 12, 13, and 16 months old), 20 control mice
(C57BL/6; 2, 3, 11, 13, and 15 months old) as well as from 12 RCS rats
(3, 11.5, and 12 months old) and the same number of RCS-control rats
(3, 5, and 13 months old). The aqueous humor from 8 eyes of each strain
and age group was pooled in one tube, immediately stored at −80°C
and thawed just before use. Unfortunately, TGF-β2 levels in aqueous
humor of ND mice could not be determined because not enough mice of
different age groups were available.
For the determination of TGF-β2 concentrations in aqueous humor we
used a double-antibody “sandwich” enzyme-linked immunosorbent assay
(ELISA; R&D Systems, Wiesbaden, Germany). This assay only detects the
activated form of TGF-β2 and does not recognize the latent form. To
activate latent TGF-β2 to the immunoreactive form, we acidified
aqueous humor (16.2 μl) by the addition of 1 N HCl. This mixture was
incubated for 10 minutes at room temperature and neutralized with 1.2 N
NaOH/0.5 M HEPES. After adding 100 μl assay diluent to each well, we
pipetted the probe or the provided standard to the microtiter ELISA
plate. After 2 hours of incubation at room temperature, each well was
washed three times with 400 μl washing buffer, and then 200 μl of
polyclonal antibody against TGF-β2 conjugated to horseradish
peroxidase was added. The incubation time was 2 hours. After washing 3
times with washing buffer the probe was incubated for 20 minutes at
room temperature in 200 μl of substrate solution (a mixture of
H2O2 and
tetramethylbenzidine). The color development was stopped by adding 50μ
l stop-solution to each well. The optical density was determined
using ELISA reader (kinetic microplate reader; Molecular Devices,
Ebersberg, Germany) set to 450 nm. For wavelength correction, readings
at 540 nm were substracted from the readings at 450 nm. For the
measurements of TGF-β2 concentrations by ELISA, each aqueous humor
sample (derived from 8 eyes per age group) was used only once, because
the entire sample volume was needed for one determination. The
determinations were performed on 3 different days with the probe
derived from 1 young (up to 11 months) and 1 old (older than 11 months)
animal group each.