3 3 diaminobenzidine dab substrate

Germ cell apoptosis was evaluated by terminal
deoxynucleotidyl transferase (TdT) enzymemediated
dUTP nick end labeling (TUNEL) assay kit
(Roche, Germany). Briefly, 5-μm thick paraffinembedded
sections were microwave-pretreated in
10 mM citrate buffer (Merck, Germany, pH=6.0)
for 10 minutes. Sections were incubated with
blocking solution (3% H₂O₂ in methanol, Merck,
Germany) for 10 minutes, then were washed with
phosphate-buffered saline (PBS, Merck, Germany).
The specimens were incubated with TUNEL
reaction mixture (TdT and nucleotide mixtures in
reaction buffer) at 37˚C for 60 minutes. Finally
the slides were stained with converter-POD (antifluorescein
antibody, Fab fragment from sheep,
conjugated with horse-radish peroxidase-POD) for
30 minutes.
The 3, 3΄- Diaminobenzidine (DAB) substrate
(Roche, Germany) was applied for color development.
TUNEL positive cells exhibited a brown nuclear
stain. In each group, the number of stained
cells was counted in 100 seminiferous tubules.
The number of stained germ cells was counted.
Apoptotic index-1 (AI-1) was defined as the number
of apoptotic TUNEL-positive cells per 100 tubules
and apoptotic index-2 (AI-2) as the number
of tubules containing apoptotic cells per 100 tubules.
All of measurements were performed by an
expert technician who was blinded to the experiment procedure.

Polyclonal anti-GPCMV IE1/2 and anti-gB antibodies^{13 (link)} and monoclonal antibody against a GPCMV early antigen g-1^{21 (link)} were used for the immunological detection of GPCMV products. Immunostaining of infected cells in the wells of culture plates was performed as follows. Infected cells were fixed with 3.7% formalin, treated with 0.05% TritonX-100 in phosphate-buffered saline (PBS), rinsed with PBS, and incubated with a dilution of g-1, anti-GPCMV IE1/2 or anti-gB antibodies. After washing three times with PBS, the cells were incubated with horseradish peroxidase-conjugated anti-rabbit or mouse immunoglobulin G antibody (Histofine Simple Stain MAXPO, Nichirei, Japan) at 37 °C for 1 hr. Positive cells were visualized with 3,3′-diaminobenzidine (DAB substrate, Roche).

Four micrometer thick paraffin sections were deparaffinized in xylene and sequentially rehydrated using a graded series of ethanol. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide. After rinsing in water for 15 min, the sections were microwave-treated with pre-warmed citrate buffer (10 mM citric acid, pH 6.0) for 15 min, cooled down to room temperature (RT) for 30 min, and blocked with 5% cosmic calf serum (CCS, HyClone, UT, USA) for 1 h. The sections were incubated with cleaved Caspase-3 antibody (1:100) overnight at 4 °C. Then, the sections were incubated with the appropriate secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) for 15 min at RT. Immunoreactivity was detected with 3,3′-diaminobenzidine (DAB) substrate (Roche, Mannheim, Germany) for 5 min and the samples were washed with 1× phosphate-buffered saline (PBS, Gibco, Grand island, NY, USA) for 10 min. The sections were then viewed by using microscopy (magnification: ×200) (OLYMPUS Microscope, Tokyo, Japan).