Two photon microscopy

PC3 cells were plated onto 12-mm diameter round glass cover slips in a six-well plate. After treatment, cells were washed three times with PBS and fixed with 3.7% paraformaldehyde for 10 min at 37°C. Fixed cells were rinsed in PBS, sequentially permeabilized with 0.2% Triton X-100 on ice for 10 min, and washed with PBS at room temperature. After incubation for 60 min in PBS/1% BSA, cells were washed in PBS/0.05% Triton X-100 and incubated with optimal dilutions of anti-Bax, anti-Tim23 primary antibodies and Hoechst for 60 min at room temperature (primary antibody IgG was diluted by PBS/0.05% Triton X-100 and 1% BSA). After three washes with PBS/0.05% Triton X-100, cells were incubated with the secondary antibodies for 60 min at room temperature: fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG (1:50) and Phycoerythrin (PE) conjugated donkey anti-mouse IgG (1:200). Cells were washed three times with PBS. Cells on cover slips were mounted with a slow-fade, anti-fade reagent onto glass slides and were observed under two-photon microscopy (Carl Zeiss, Thornwood, NY).

For analysis of thymic medulla and cortex by immunohistology, thymi from GCV treated TK⁻ and TK⁺ mice were fixed in 4% formalin and embedded in paraffin blocks. Sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined by light microscopy. For immunofluorescence, serial sections (5 μm) from OCT-embedded frozen tissues or primary cultured cells were fixed in cold acetone or 4% polyoxymethylene and blocked in PBS/1% BSA, washed in PBS/0.05% Tween and incubated with optimal dilutions of fluorochrome-conjugated antibodies: Alexa Fluor® 488 anti-I-A/I-E, anti-CD31-PE (Biolegend), anti-CD11c-PE, anti-CD11b-FTIC (BD Pharmingen), and anti-F4/80-PE (eBioscience), or with first Abs: anti-cytokeratin 5, anti-FSP1, anti-ER-TR7 (Abcam), anti-cytokeratin 8 (Tromal-1; Developmental Studies Hybridoma Bank), Biotinylated UEA-1, anti-vimentin (BD Pharmingen), anti-α-SMA, anti-Pan-CK (Sigma, Cat no. C5992), anti-CD140a/PDGFRα (R&D Systems) and anti-MTS15 Ab for 2 h at room temperature before washing and incubating with secondary reagents: Alexa Fluor® 546 Goat anti-Rabbit/mouse IgG (H+L), Alexa Fluor® 488 Goat anti-Rat IgG (H+L) (Invitrogen), Dylight 488 Goat anti-Rabbit/mouse IgG (ZSGB-Bio) and streptavidin-PE (BD PharMingen). Control slides were incubated with isotype-matched Ig. Images were acquired with a two-photon microscopy (Carl Zeiss, Inc.).