Journal: Frontiers in Cellular Neuroscience
Article Title: Disruption of the Microglial ADP Receptor P2Y13 Enhances Adult Hippocampal Neurogenesis
Figure Lengend Snippet: Lack of colocalization of P2ry13 mRNA with other cell types of the DG (young adults). (A–E) Details of DG showing double labeling using FISH for P2ry13 (red) and immunocytochemistry for various cell markers (green); overlay (left), immunolabeling (right; all WT animals). (A) Glial fibrillary acidic protein (GFAP), (B) Sox2, (C) DCX (weak immunolabeling due to experimental conditions required for FISH) (D) NeuN. (E) Triple labeling for somatostatin (green, St), parvalbumin (white PV) and for P2ry13 (red). The GCL is outlined with dashed lines. Arrows, cell bodies labeled for P2ry13 mRNA; arrow head, P2ry13 -negative astrocyte. (F) Simplified schematic of principal cell types along the hippocampal neurogenesis pathway. Lineage markers used in this study and their cellular location are indicated by bars. gcl, granule cell layer; mol, molecular layer; sgz, subgranular zone. Scale bars, 25 μm.
Article Snippet: Single- or two-color FISH combined with immunohistochemistry was performed as described (Ishii et al., ) with slight modifications: (1) hybridization step: slices were incubated in 12 μg/ml of proteinase K solution at 37°C for 16 min; (2) hybridization of P2ry13 and Cx3cr1 riboprobes labeled with digoxigenin and fluorescein, respectively, was performed at 65°C overnight in the hybridization solution (Parrilla et al., ); and (3) detection step: antibodies against digoxigenin-alkaline phosphatase (11093274910, Roche) and fluorescein horseradish peroxidase (NEF710, PerkinElmer) were incubated together with the following primary antibodies: anti-Iba1, anti-GFAP, anti-SRY (sex determining region Y)-box 2 (Sox2; Sc-17320, 1:200, Santa Cruz), anti-DCX, anti-NeuN (ABN78, 1:500, Millipore), anti-somatostatin (AB5494, 1:200, Millipore) and/or anti-parvalbumin (PV27, 1:200, Swant).
Techniques: Labeling, Fluorescence In Situ Hybridization, Immunocytochemistry, Immunolabeling