A11006

Adult rat brains were resected three weeks after AAV2-Cre injection and promptly sectioned at 250 μm using a vibratome (VT 1000S, Leica). Tissue slices were fixed with 4% paraformaldehyde, reacted with anti-Cre antibody (1:500, MAB3120, Millipore) and anti-DsRed antibody (1:500, #632496, Clontech), followed by secondary antibodies conjugated with Alexa Fluor 488 (1:200, A11006, Molecular Probes, Eugene, OR, USA) and Alexa Fluor 546 (1:200, Z25004, Molecular Probes, Eugene, OR, USA), respectively. Immunoreactivity was assessed by fluorescent microscopy (Axiovert200, Carl Zeiss) or confocal microscopy (LSM510META, Carl Zeiss) or FV1200, Olympus). For the detection of each fluorescence substrate, following optical combinations were used: a 488 nm argon laser and 525 ± 25 nm bandpass filter for Alexa Fluor 488, a 543 nm HeNe laser and 590 ± 25 nm bandpass filter for Alexa Fluor 546.
To evaluate the efficiency of in vivo recombination, brain slices (16 μm) were prepared from AAV2-Cre injected brain tissue and immunostained against Cre and tdTomato. In each slice, the immunolebeled cells were counted in an unintentional visual field (0.088 mm²) selected from a brain slice of the AAV-injected region.

Mice were deeply anesthetized with isoflurane and transcardially perfused with ice-cold PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were removed, post-fixed in PFA overnight, and equilibrated in a 30% sucrose solution. 40 µm sections were obtained using a frozen sliding microtome (American Optical). Slices were placed in cryoprotectant (25% glycerol, 30% ethylene glycol, 45% PBS, pH adjusted to 6.7 with HCl) for long-term storage. Before immunostaining, slices were washed in PBS to remove the cryoprotectant and then blocked and permeabilized for one hour at room temperature with 0.3% Triton X-100 (Sigma) and 3% normal goat serum (NGS) in PBS. We incubated slices for ~48 hr at 4^o C with a primary antibody for either parvalbumin (Swant PV27, 1:1000) or somatostatin (Millipore MAB354, 1:50) in PBS with 0.3% Triton X-100% and 3% NGS. Slices were then washed with PBS and incubated with a secondary antibody in PBS with 0.3% Triton X-100% and 1% NGS: Alexa Fluor 488 goat anti-rat for PV staining (Molecular Probes, A11006) or goat anti-rabbit for SST staining (Molecular Probes, A11034). Slices were washed, labeled with DAPI (1:50,000; Fisher Scientific), and cover-slipped using polyvinyl alcohol mounting medium with DABCO (Sigma 10981). Imaging was performed using an upright epi-fluorescence microscope (Nikon Instruments).

Liver sections were cut at 12 μm thickness with a cryostat (Leica). For immunofluorescent staining, sections underwent antigen retrieval in 10 mM sodium citrate buffer. Sections were blocked for 1 hour in 5% BSA in PBS at RT. Sections were incubated with primary antibody (anti-HA 1:200 in 5% BSA (#ROAHAHA, Sigma)) for 16 hours at 4 ^°C. Sections were the incubated with secondary antibody for 2 hours at RT (Alexa-fluor anti-Rat/594 1:200 in 5% BSA (#A-11007, Molecular Probes)) and mounted with Vectashield hard set mounting medium with DAPI.
Live fibroblasts on coverslips were incubated with MitoTracker Red CMXRos (100 nM for 30 minutes) (Molecular Probes) then fixed with 4% PFA. Fibroblasts were permeabilized with ice-cold methanol and blocked for 1 hour in 5% BSA in PBS at RT. Coverslips were incubated with primary antibody (anti-HA 1:200 in 5% BSA (#ROAHAHA, Sigma)) for 16 hours at 4 ^°C. Coverslips were the incubated with secondary antibody for 2 hours at RT (Alexa-fluor anti-Rat/488 1:200 in 5% BSA (#A-11006, Molecular Probes)) and mounted with Vectashield hard set mounting medium with DAPI. Images were captured using a Zeiss LSM710 confocal microscope.