Mouse monoclonal anti cr

For quantitative analysis of GABA-expressing neurons, serial coronal sections (7 µm) from the location of the open defect (exposed area) of the lumbar cord in the SBA chicks, and from a similar location in the normal chicks, were stained with a rabbit polyclonal anti-GABA antibody (1 : 1500; Sigma). The tissues were observed under a Nikon Eclipse E800 light microscope, and images were acquired using a charge-coupled device (CCD) camera attached to the microscope (Nikon Digital Sight DS-L2). For analysis of GABAexpressing neurons, all GABA-immunopositive cells with a rounded profile in superficial dorsal horn laminae I-III were considered and counted. The total num-ber of neurons in the superficial dorsal horn on each side of the spinal cord was calculated and averaged across six cross sections per chick at each age; each group comprised six chicks of each age.
For quantitative analysis of PV-, CB-, and CR-expressing neurons, serial coronal sections were stained with mouse monoclonal anti-PV (1 : 500; Sigma), mouse monoclonal anti-CB, or mouse monoclonal anti-CR (1 : 500; SWANT) antibody, and PV-, CB-, and CR-immunopositive cells were enumerated as described above.

Immunofluorescence staining was performed as described previously [17] . For double immunofluorescence staining, the sections were incubated for 60 h at 4°C in a solution containing rabbit polyclonal anti-GABA (1 : 3000; Sigma) plus mouse monoclonal anti-CB or mouse monoclonal anti-CR (1 : 1000; SWANT) antibodies; and rabbit polyclonal anti-caspase 3 (1 : 1000; Bioss) plus mouse monoclonal anti-CB (1 : 1000; SWANT) antibodies. After washing in PBS, the sections were treated for 2 h at room temperature with an Alexa Fluor 546-conjugated goat anti-rabbit IgG (H + L) (1 : 1000; Invitrogen) or an Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) (1 : 1000; Invitrogen) and 4',6-diamidino-2-phenylindole (DAPI), washed with PBS, mounted with Vectashield (Vector Laboratories), and visualised using a Nikon A1 confocal microscope equipped with a 100× objective lens (Nikon).