Elite kit

The sections were washed six times in PBS to remove any residual cryoprotectant (10 min each). This was followed by an endogenous peroxidase activity block in 0.3% hydrogen peroxide solution (0.3% H₂O₂, 10% methanol, distilled water), after which the sections were washed three times in PBS (10 min each). To minimize non-specific binding, sections were incubated in 3% horse serum in PBST (PBS plus Triton X-100 at 500 μL/1L PBS) for 1 h and then incubated for 48 h with a primary anti-calbindin antibody (D-28 K, CB300, Swant, Switzerland) in 1% horse serum in PBST. Following this incubation period, sections were washed three times in PBST (10 min each) and incubated with a secondary antibody (in 1% horse serum in PBST) raised against the primary antibody molecule for 2 h (BA-2000, Vector Laboratories). This was followed by three PBST washes (10 min each) and incubation in an avidin–biotin-kit solution (Elite Kit, Vector Laboratories) for 1 h. Following four more washes (10 min each), the label was developed with the DAB kit according to the manufacturer’s instructions (DAB Substrate Kit, Vector Laboratories). All incubations took place at room temperature and, except for the final incubation, on a shaker. The sections were then mounted on gelatin-coated glass slides, air-dried and cover-slipped.

To minimize background fluorescence and fading, GFP(+) GIN and G42 cells were stained using anti-GFP immunochemistry (1°: rabbit anti-GFP, #A11122 Life Technologies, 1:10,000 dilution, 24 h, 20°C; 2°: donkey anti-rabbit Biotin-SP Affinipure #711-065-0152, Jackson Immunoresearch Laboratories, 1:100 dilution, 1 h, 20°C) followed by avidin-biotin-peroxidase reaction (Elite Kit, Vector Laboratories) using 3,3′-diaminobenzidine (DAB). PV expressing cells were immunostained using rabbit anti-parvalbumin (1°: PV27, Swant, 1:1000, 48 h, 4°C; 2°: donkey anti-rabbit Alexa-fluor-488, #A21206 Life Technologies, 1:500, 3 h, 20°C). SST expressing cells were immunostained using rabbit anti-somatostatin (1°: Ab20067 Immunostar, 1:500, 48 h, 4°C; 2°: donkey anti-rabbit Alexa-fluor-488, #A21206 Life Technologies, 1:500, 3 h, 20°C). Prior to staining, tissue was blocked in 10% normal donkey serum (1 h). Specificity controls for the secondary antibodies were performed by excluding the 1° antibody in a small number of sections (n = 4). Only faint neuropil fluorescence was visible and no cell bodies were stained.

Animals were perfused transcardially with ice-cold saline and brains were removed and immersed into 4% PFA in PBS. After dehydration in 30% sucrose in PBS, coronal sections (30 μm in thickness, one in tenth series) were prepared with a sliding microtome (Leica). For immunofluorescence staining, free floating sections were incubated with the following primary antibodies: goat anti-DCX (sc-8066, Santa Cruz; 1:100), rabbit anti-Ki67 (s2532, Sigma; 1:500), rabbit anti-Iba1 (019-19741, Wako; 1:1,000), rabbit anti-NeuN (MABN140, Millipore; 1:1,000), rabbit anti-MAP2 (4542, Cell Signaling; 1:500), rabbit anti-AQP4 (AQP-004, Alomone labs; 1:1,000) followed by incubation with appropriate secondary antibodies conjugated with 488 (Vector Laboratories, 1:500) or Cy3 or 594 (Jackson, 1:500). For immunohistochemical staining, brain sections were incubated with 3% H₂O₂ in methanol, to quench endogenous peroxidase activity followed by incubation with primary antibodies: mouse anti-GFAP (G3893, Sigma; 1:2,000) and rabbit anti-vimentin (ab92547, abcam; 1:1,000). After washing, sections were incubated with biotinylated goat anti-mouse or rabbit (1:200, Vector Laboratories). Binding of the antibodies was detected with the Elite kit (Vector Laboratories) with diaminobenzidine (Sigma) and H₂O₂ for development. All immunostaining analyses were done blindly.

Coronal brain sections and cocultured bEnd.3 cells were rinsed twice in PBS, incubated in 0.2 % Triton X-100 for 30 minutes at room temperature, and rinsed three times with 0.5 % bovine serum albumin (BSA) in 1× PBS for blocking. After blocking, they were incubated overnight at 4 °C with primary antibody (anti-TH, anti-P-gp, anti-MPO, or anti-EBA), rinsed three times in 1× PBS containing 0.5 % BSA (10 minutes/rinse), and incubated with the appropriate biotinylated secondary antibody and avidin–biotin complex (Elite Kit; Vector Laboratories, Burlingame, CA, USA) for 1 hour at room temperature. Bound antibodies were visualized by incubating with 0.05 % diaminobenzidine–HCl (DAB) and 0.003 % hydrogen peroxide in 1× PBS. Brain sections were rinsed with 1× PBS for DAB inhibition. Immunostained cells were analyzed by bright-field microscopy.