Pv235

Following recording, cells were resealed by obtaining outside-out patches and slices immersion-fixed in 4% paraformaldehyde (PFA) in 0.1 m phosphate buffer (PB; pH 7.4) at 4°C for 24–48 h. Slices were then transferred to fresh PB and stored for a maximum of three weeks. Before immunohistochemical processing, slices were rinsed in PB, followed by PBS (0.9% NaCl) and blocked in PBS containing 10% normal goat serum (NGS), 0.3% Triton X-100 and 0.05% NaN₃ for 1 h. Slices were then incubated with a monoclonal mouse antibody raised against PV (1:5000, PV-235, Swant) in PBS containing 5% NGS, 0.3% Triton X-100 and 0.05% NaN₃ for 72 h at 4°C. Slices were then rinsed in PBS and a fluorescent-conjugated secondary antibody was applied (goat anti-mouse IgG, Alexa Fluor-405, 1:1000, Invitrogen) in combination with fluorescent-conjugated streptavidin (Alexa Fluor-647, 1:1000, Invitrogen), in a PBS solution containing 3% NGS, 0.1% Triton X-100 and 0.05% NaN₃ for 24 h at 4°C. Slices were rinsed in PBS and then desalted in PB before being mounted in on glass slides (Fluoromount-G, Southern Biotech) with a 300-μm-thick agar spacer, cover-slipped, sealed and stored at 4°C before imaging.

For identification of cells following recordings, tissue was fixed in 4% paraformaldehyde overnight at 4^oC. Slices were washed with PBS three times, 20 min each, and incubated in Streptavidin-Alexa 488 (S11223) or Alexa 647 (1:500, S32357, Invitrogen) and DAPI (1:1000, in 0.3% PBST) overnight at 4°C. Slices were washed in 0.3% PBST four times and mounted on glass slides with Mowiol 4-88. Mcherry fluorescence in the MEC was examined with confocal imaging and those animals where virus injection missed MEC L5 were excluded.
For staining of the interneuron marker Parvalbumin (PV), slices were prepared as described above, then incubated in primary antibody solution containing mouse anti-PV (1:1000, PV 235, Swant) and 5% NGS in 0.3% PBST for 48 hr at 4°C. Slices were then washed and incubated in secondary antibody solution with Alexa-conjugated Streptavidin and DAPI prepared in 0.3% PBST and mounted on glass slides after overnight incubation.
A Zeiss LSM800 microscope was used for image acquisition of the slices. Pinhole was set to 1 Airy Unit. Objectives used include ×10 (air), ×20 (air), and ×40 (oil) to image the hippocampal formation, morphology of biocytin filled cells, and immunolabelling of interneurons, respectively.

Mice were perfused with PBS, followed by 4% (w/v) paraformaldehyde in PBS (pH 7.4). Brains were harvested and fixed with 4% paraformaldehyde overnight, transferred to 30% (w/v) sucrose in PBS, and then cryosectioned into 40 μm thick slices. For immunohistochemistry, brain slices were permeabilized with 0.2% Triton X-100 in PBS for 10 min and transferred to blocking buffer (5% donkey serum, 2% BSA and 0.2% Triton X-100 in PBS) for 1 h at room temperature. Next, the sections were incubated with primary antibody for parvalbumin (1:1000 diluted in blocking buffer, PV-235, Swant) overnight at 4 °C. The sections were subsequently incubated with appropriate secondary antibodies (1:500 diluted in blocking buffer, anti-streptavidin Alexa Fluor 488, anti-mouse Alexa Fluor 555; Invitrogen) for 2 h at room temperature. All sections were then stained with DAPI (1:5000 diluted with 0.2% Triton X-100 in PBS, Sigma-Aldrich) and mounted with Fluorsafe (Merck Millipore). Images were captured using an LSM 710 confocal microscope (Zeiss).

Slices were permeabilized in blocking buffer (10% normal goat serum and 1% TritonX-100 in PBS) for 2 h at room temperature and washed three times with PBS. Antibodies were prepared in 1% blocking buffer: biotin-conjugated anti-NeuN at 1:1000 (Merck, MAB377B); mouse anti-parvalbumin at 1:2000 (Swant, PV 235); rabbit anti-calbindin at 1:2000 (Swant, CB38); rabbit anti-Olig2 at 1:1000 (Merck, AB9610); streptavidin Alexa Fluor 647, goat anti-mouse Alexa Fluor 647, and goat anti-rabbit Alexa Fluor 555 at 1:500, (Thermo Fisher, S32357, A21235, A21428). Slices were incubated in primary antibodies overnight at 4 °C and washed three times in PBS. Slices were then incubated in secondary antibodies for 2 h at room temperature and washed three times in PBS. Nuclei were stained with DAPI.

Free-floating brain slices were rinsed three times in PBS and digested with chondroitinase ABC (C3667, Sigma; for details, see immunohistochemistry) as enhancing step for the AB1031 aggrecan staining (AB1031, Millipore). The sections were then blocked with blocking solution (5% normal donkey serum, Jackson ImmunoResearch, UK and 0.25% Triton X-100 in PBS) for one hour at RT. The sections were incubated overnight at 4°C with a primary antibody (polyclonal rabbit anti-aggrecan 1∶1000, AB1031, Millipore; monoclonal mouse anti-chondroitin sulfate proteoglycan 1∶1000, protein core epitope, Cat-315/MAB1581, Millipore; monoclonal goat anti-parvalbumin, 1∶2500, PV235, Swant) diluted in blocking solution. Following three rinses with 2% normal donkey serum and 0.25% Triton X-100 in PBS, the sections were incubated with appropriate secondary antibodies (donkey anti-rabbit/mouse/goat antibody) conjugated with either Cy3 or DyLight 488 fluorescent dyes (Jackson ImmunoResearch, UK) at a dilution of 1∶400 in blocking solution for 90 minutes at RT. Fluorescent dyes were imaged using a confocal laser-scanning microscope (Zeiss LSM 510, Jena, Germany).