Image pro plus image analysis software system

On the 7th day after the establishment of the CCI rat model, five rats were randomly chosen from each group and executed under chloral hydrate anesthesia. L4 to L6 dorsal spinal cord tissues were collected and stored in 100g/L paraformaldehyde solution and immobilized at room temperature (RT) for 24 hours. Coronal sections (3~4 μM thick) of tissues were placed in an oven for 4~6 hours. Then, the sections were cleaned with PBS three times (2 min each time) and maintained with the rabbit anti-GFAP antibody (Abcam, ab7260, 1: 500, MA, USA) in a refrigerator overnight at 4° C. After washes, the horseradish peroxidase-labeled goat anti-rabbit IgG (1:800) was added and incubated at 37° C for two hours. Afterward, the sections were cleaned, and DAB was adopted for color development. The percentage of area occupied by GFAP-positive astrocytes in the L4 to L6 dorsal spinal cord tissue was assessed with the Image-Pro Plus image analysis software system (Media Cybernetics, MD, USA).

Spinal cord tissues (L4-L5) were routinely paraffin-embedded, sectioned, dewaxed and hydrated before antigen repair with proteinase K (0.2 mg/mL) and 10 m/Vl sodium citrate solution (pH 6.0). The sections were closed with 2% bovine serum albumin (BSA) for 30 minutes at RT and then kept with rat anti-GFAP monoclonal antibody (1:500; Millipore, USA) and rabbit anti-lbal monoclonal antibody (1:300; Wako Pure Chemical Industries, Ltd Japan), respectively, for 24 hours at 4°C. Next, they were maintained with the HRP-labeled secondary antibody for 2 hours and developed with diaminobiphenyl (DAB) amine solution. The number of GFAP-positive astrocytes and Ibal-positive microglia in spinal cord tissues was estimated by utilizing the Image-Pro Plus image analysis software system (Media Cybernetics, USA) [25 (link)].