Tcs speii

Under deep anesthesia by intraperitoneal injection of sodium pentobarbital (100 mg/kg), the animal was perfused with normal saline followed by 4 % paraformaldehyde in 0.1 M PBS, pH 7.2–7.4, for 20 min. Then L4 and/or L5 lumbar segment were dissected out and post-fixed in the same fixative. The embedded blocks were sectioned as 30-μm thick and processed for immunofluorescence assay. Sections from each group (five mice in each group) were incubated with primary antibody (IBA-1, 1:200; c-fos, 1:200; CGRP, 1:200; NeuN, 1:300). Then the free-floating sections were washed with PBS, and incubated with the secondary antibody (1:300; Jackson Laboratories, USA) for 2 h at room temperature. After being washed three times with PBS, the samples were investigated with a confocal microscope (Leica TCS SPEII, Leica Biosystems, Wetzlar, Germany) for morphologic details. Images were randomly coded and transferred to a computer for further analysis.

After anesthesia by intraperitoneal injection of sodium pentobarbital (100 mg/kg), the animal was perfused with normal saline followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.2–7.4, for 20 min. Then, L4 and/or L5 lumbar segment were dissected out and post-fixed in the same fixative. The embedded blocks were sectioned as 30 μm thick and processed for immunofluorescence assay. Sections from each group (five mice in each group) were incubated with primary antibody (IBA-1, 1:200; c-fos, 1:200; CGRP, 1:200). Then, the free-floating sections were washed with PBS and incubated with the secondary antibody (1:300; Jackson Laboratories, USA) for 2 h at room temperature. After using PBS to wash three times, the samples were investigated with a confocal microscope (Leica TCS SPEII, Leica Biosystems, Wetzlar, Germany) for morphologic details. Images were randomly coded and transferred to a computer for analysis.