Computational protocol: Hypocellularity in the Murine Model for Down Syndrome Ts65Dn Is Not Affected by Adult Neurogenesis

Similar protocols

Protocol publication

[…] Tissue was processed “free-floating” for immunofluorescence as follows. Briefly, sections were incubated with citrate buffer (0.01M, pH 6.0) for 1 min at 100°C. After this, sections were treated for 1 h with 5% normal donkey serum (NDS) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in PBS with 0.2% Triton- X100 (Sigma-Aldrich, St Louis, MO, USA) and were incubated overnight at room temperature in polyclonal rabbit IgG anti-cleaved caspase-3 (1:500, Cell Signaling, 9661) antibody. After washing, sections were incubated for 2 h with donkey anti-rabbit IgG conjugated with DL549 (1:200, Jackson Immunoresearch) for 2 h. After washing, sections were mounted on slides using Dako fluorescent medium (Dako North America, California). The sections were counterstained with 4′, 6-Diamidino-2-phenylindole (DAPI) (dilution 1/500) (Sigma-Aldrich, St Louis, MO, USA) to identify cellular nuclei. The analysis of sections was performed using a confocal microscope (Leica TSC-SPE) using 40X objective. Serial stacks of the different regions (granule layer of dentate gyrus, RMS and olfactory bulb) were analyzed using ImageJ software. Interval between z planes was 1.15 μm. [...] In order to characterize the phenotype of the newly born cells in the dentate gyrus and the olfactory bulb, we have performed a triple immunolabeling using antibodies against 5′BrdU, NeuN (neurons) and GFAP (astrocytes). The triple labeling was performed following the “free-floating” procedure described previously with some changes.Sections were incubated in PBS during 60 min to 60°C. After cooling down, sections were incubated with HCl 2N for 30 min. After washing, sections were incubated with a blocking solution containing 10% NDS for 1 h and overnight with a mix of monoclonal rat IgG anti-5′BrdU (1:1000, Inmunological Direct, OBT0030), polyclonal rabbit IgG anti-GFAP (1:500 Sigma Aldrich, G9269) and monoclonal mouse IgG anti-NeuN (1:100, Millipore, MAB377) antibodies. After washing, sections were incubated with a mix of secondary antibodies containing: Donkey anti-rat IgG conjugated with Alexa 488 (1:200, Molecular Probes), donkey anti-rabbit IgG conjugated with DL649 (1:200, Jackson Immunoresearch) and donkey anti-mouse IgG conjugated with DL549 (1:200, Jackson Immunoresearch) for 2 h. After washing, sections were mounted on slides using Dako fluorescent medium (Dako North America, California). The analysis of sections was performed using a confocal microscope (Leica TSC-SPE) using 40X objective. Stacks (z-step 1.15 μm) of the different regions (granule layer of dentate gyrus and olfactory bulb) and were analyzed using ImageJ software. A minimum of 50 cells were analyzed for each animal. Percentages for every marker were obtained and mean for each group and region was determined and analyzed using SPSS software. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Movement Disorders