C4742 95 camera

Lentivirus transduced, floating neural precursor aggregates were gently trypsinized to single cells and left to attach 1 h at 37 °C to poly-L-lysine (200 μg/ml) coated SuperFrost Plus microscope slides (Menzel-Glaser). Here they were fixed by 4% PFA for 20 min at 4 °C, washed three times in 1X PBS and processed for immunofluorescence. Fixed-cryopreserved brains were sliced at 16 μm, tissue slices were allowed to dry at least one hour at RT and processed for immunofluorescence.
In all cases, immunofluorescence was performed as previously described45 (link). The following primary antibodies were used: anti-Tubb3 (mouse clone Tuj1, Covance #MMS-435P, 1:1000); anti-GFP (chicken polyclonal, Abcam ab13970, 1:400); anti-Foxg1 (rabbit polyclonal, 1:20041 (link)). Secondary antibodies were conjugates of Alexa Fluor 488 and 594 (Invitrogen), used at 1:600. Cell nuclei were counterstained with DAPI (4′, 6′-diamidino-2-phenylindole).
Tubb3 immunofluorescences were photographed on a Nikon Eclipse TS100 fluorescence microscope equipped with a DS-2MBWC digital microscope camera with a 20X objective. Immunoprofiled brain sections were photographed on a Nikon TI-E microscope, equipped with 20X or 40X objectives and a Hamamatsu C4742–95 camera. All images were processed using Adobe 9.0.2 Photoshop 2 CS2 software and ImageJ.

The immunofluorescence technique was performed as previously described (Gerig and Celio, 2007 (link); Mészár et al., 2012 (link)). In short, CTR, HET, and KO mice (n = 3/genotype) were perfused with 0.9% NaCl, followed by 4% paraformaldehyde, diluted in 0.1M phosphate buffered saline, pH 7.3. The brains were dissected and immersed in 30% sucrose for one night, and then frozen on dry ice. Eighty micrometers of thin cryomobile (Reichert Jung) sections were prepared and incubated in parallel in 24-well plates with the primary antiserum against VGlut2 (Synaptic Systems, Göttingen, Germany), diluted 1:10,000 to 1:20,000. The efficacy and specificity of this antiserum against VGlut2 was established by immunoblotting experiments (see section Western Blot of Brain Extracts). Incubation with the biotinylated secondary antibody was followed by exposure to streptavidine-Cy3 (Alexa 550). The sections were mounted on glass slides for histological inspection in either a Leica 6,000 epifluorescence microscope (equipped with a Hamamatsu C4742-95 camera) or a digital slide-scanner (Nanozoomer, Hamamatsu).

Cells were loaded with 2 μM Fura2-AM (Molecular Probes) for 30 minutes and allowed to recover in standard extracellular solution for a further 30 minutes before experiments. All recordings were performed at room temperature. The cells were superfused with standard extracellular solution for at least 5 minutes before beginning recordings. Ratiometric measurements of intracellular calcium [Ca²⁺]i were made using a Cairn Dual OptoLED (excitation wavelengths: 350 and 380 nm) with a Hamamatsu C4742-95 camera and Simple PCI 6 software (Hamamatsu). Background subtraction and ratio calculations (350/380 nm) were performed within the software. Standard extracellular solution contained the following (mM): NaCl (140); KCl (4); CaCl₂ (2); HEPES (10); NaOH (4.54) and glucose (5). For high potassium extracellular solution, the concentration of KCl was increased to 40 mM and NaCl was reduced to 104 mM. Both solutions were adjusted to pH 7.4 at room temperature using NaOH. We tested three fields of cells for each compound. To avoid desensitisation mechanisms, only one stimulus was applied per coverslip, unless the cells failed to respond, in which case another drug was tested on the same coverslip.

Embryos were harvested in cold PBS and cryopreserved as described previously (see Immunofluorescence Antibody Staining). Anti‐active Caspase‐3 primary antibody (Promega Corp.) was applied at 1 : 200 in 1% NDS/TSP for 1 h 37 °C with rocking. After washing, fluorescent secondary antibody, AlexaFluor™647 (Invitrogen‐ThermoFisher Scientific) was applied, also at a dilution of 1 : 200. Slides were then washed and counterstained with DAPI at (1 : 5000) for 10 min, followed by coverslipping. Three pairs of embryos (control and mutant) were analyzed for each time point (E11.5–E13.5). Palatal sections were categorized as anterior, middle or posterior depending on their location and stereotypical morphology. Fluorescent images of palatal sections were obtained using a Hamamatsu C4742‐95 camera (Hamamatsu Corp.), mounted on a Nikon Eclipse TE200 microscope (Nikon Corp.). Quantification of the percentage of Caspase‐three positive cells vs. total number of cells present in each palatal domain was performed using algorithms generated with matlab software, as described above for the Cell Proliferation Assay. The total number of cells counted on each section was 400–700.

Capsule thickness was measured using the FITC-dextran zone of exclusion method, as previously described, with minor modifications^{40 (link)}. Exponential phase cultures were centrifuged at 3000 g for 10 min, and the pellet re-suspended in PBS. 10 µl of bacterial suspension was mixed with 1 µl of 2000 kDa FITC-dextran (Sigma-Aldrich) and pipetted onto a microscope slide. The Nikon Eclipse 80i fluorescence microscope (100 × magnification) was used to view the slides and photographs were taken using a Hamamatsu C4742-95 camera. ImageJ was used to determine the zone of exclusion (area in pixels), a value proportional to capsular thickness.