Twenty-one day old CD-1 male mice (Charles River Breeding Laboratories, Raleigh, NC, USA) were randomly assigned to experimental groups and administered a single intraperitoneal (i.p.) injection of either 2 mg/kg trimethyltin hydroxide (TMT, originally obtained from Alfa Products, Danvers, MA, USA) or saline (injection vol., 2 ml/kg). Each animal was injected with bromodeoxyuridine (BrdU; 50 mg/kg i.p.) at the time of TMT dosing, and twice daily for 3 days (Takagi et al., 1999
). Animals were randomly assigned to time points for examination and individually housed in a dual corridor, semi-barrier animal facility at ambient temperature (21 ± 2o
C), humidity (50% ± 10%) under a 12-h light/dark cycle. Autoclaved NIH 31 rodent chow and deionized, reverse osmotic-treated water were available ad libitum
. All procedures were performed in compliance with an animal protocol approved by the NIEHS/NIH Animal Care and Use Committee.
At 72 h and 1, 2 or 4 weeks following saline or TMT injection, animals (15 per group) were lightly anesthesized by CO2, decapitated, the brains quickly excised, bisected on midsaggital plane and immersion fixed in 4% paraformaldehyde/0.1M PBS, pH 7.2, overnight at room temperature (RT). Brains were rinsed with PBS, dehydrated in ethanol, embedded in paraffin, and 8-μm sections cut through the hippocampus. Sections were cleared and rehydrated, subjected to heat-induced epitope retrieval (HIER) in 0.01 M citrate buffer (pH 6.0) using a decloaking chamber (Biocare Medical, Walnut Creek, CA) for 3 min, rinsed, and maintained at RT for 20 min. Routine hematoxylin and eosin (H&E) staining visualized tissue cellularity. Cell counts of neurons were recorded on 10 randomly selected hippocampal sections per animal. Using an ocular counting grid the number of viable neurons within 3 identified grid locations of the dentate (total area per grid section 0.105 mm2) was measured at 72 h and 4 weeks post-TMT.
For BrdU histochemistry, 3–5 sections per animal were incubated in 2N HCl (37o
C, 30 min), trypsinized (0.01% trypsin in 0.5M Tris-HCl; 37o
C, 3 min), endogenous peroxidase activity quenched with 3% H2
(10 min), and endogenous biotin activity quenched using a commercial avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA, USA). BrdU incorporation was localized by a 1 h incubation with 1:400 rat monoclonal anti-BrdU (Accurate Chemical and Scientific Corp, Westbury, NY, USA), Vectastain Elite®
ABC Rat IgG, and DAB chromagen (Vector Labs). Immunofluoresence for BrdU was detected with either AlexaFluor®
488 or 594. Proliferation was also identified with a mouse monoclonal antiserum for Ki-67 (1:100; NCL-L-Ki67-MM1, Nova Castra, Newcastle upon Tyne, UK) (Kee et al., 2002
), a mouse-on-mouse peroxidase kit (MOM™
, Vector Labs) as visualized by DAB. Apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ
end-labeling (TUNEL) using a commercially available kit (Intergen, Purchase, NY, USA).
Astrocytes were identified using an Alexa Fluor®
594 conjugated mouse monoclonal anti-glial fibrillary acidic protein (GFAP; 1:200; 1.5 h, RT; Molecular Probes, Eugene, OR, USA). Microglia were identified by lectin binding (Griffonia (Bandeiraea) simplicifolia
, IB4). Briefly, sections were pre-incubated in PBS containing cations (0.1 mM CaCl2
) and 0.1% Triton X-100, for 30 min RT, incubated with IB4 (1:200; 1.5 h, RT; Molecular Probes) conjugated to AlexaFluor®
594. Mature neurons were identified with immunostaining for NeuN (1:100; Chemicon International, Temecula, CA, USA) detected with anti-mouse IgG or streptavidin AlexaFluor®
488. As additional markers of neurogenesis (Kempermann et al., 2003
; Jin et al., 2001
), mouse monoclonal anti-nestin (1:100; Chemicon) was detected using a MOM™
kit and goat anti-mouse IgG-AlexaFluor®
594 (Molecular Probes) and goat polyclonal anti-doublecortin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) using a Vectastain Elite®
ABC goat IgG kit and Novared®
(Vector) chromagen, and counterstained with hematoxylin QS.
Light and fluorescence microscopy was performed with a Leica DMRBE microscope (Wetzlar, Germany) equipped with differential interference contrast (DIC/Nomarski) optics, epifluorescence, and motorized Z-control. Digital images were acquired using a SpotRT™ cooled, charged-couple device camera (Diagnostic Instruments, Sterling Heights, MI) under the control of Metamorph™ software (Universal Imaging Co., Downingtown, PA, USA).