Retiga 2000r camera

At the end stage (19 weeks) or at 15 weeks of life, the SOD1(G93A) mice (n = 5 per group) were deeply anesthetized and transcardially perfused with PBS 0.1 M followed by paraformaldehyde 4%. The spinal cord was dissected out and 2 h of post-fixation was performed. The lumbar tract was soaked in 30% sucrose, included in OCT and serially cut at 15 µm with cryostat apparatus. The sections were mounted on Surgipath^® Apex™ Superior Adhesive Slides (3800080E, Leica Biosystems Italia, Milan, Italy).
For Nissl staining, the slides were air-dried, and then hydrated with H₂O for 30 sec. The sections were stained with 0.2% cresyl violet solution for 8 min and gradually placed into increasing concentrations of ethanol, cleared with xylene, mounted with Entelan and covered with a cover glass. The MN of the ventral horn in the lumbar spinal cord tract (lateral and medial MN of L1-L5 segments) were counted blinded every 100 µm (every 6/7 slide) by the operator using a computer-assisted microscope (Olympus BX6 with Retiga 2000R camera, Center Valley, PA) with the Stereoinvestigator software (MicroBrightField, Williston, VT, USA) at 40× magnification. Cells with nucleoli on the plane of focus, size and shape typical of MN were counted. The values from the sections were computed for the summation, the mean number was then computed from the average number derived from each animal.

The end stage SOD1(G93A) mice (n = 5 per group) were deeply anesthetized and transcardially perfused with PBS 0.1 M followed by paraformaldehyde (PFA) 4%. The spinal cord was dissected out and post-fixed with PFA 4% overnight. The lumbar tract was soaked in 30% sucrose, included in OCT and serially cut at 20 µm with cryostat apparatus. The sections were mounted on Surgipath®Apex™ Superior Adhesive Slides (3800080E, Leica Biosystems).
For Nissl Staining the slides were air-dried and then hydrated with H₂O for 30 sec. After the staining with 0.2% cresyl violet solution for 8 min, the sections were gradually placed into increasing concentrations of ethanol, cleared with xylene, mounted with Entelan and covered with cover glass. The MNs of the lumbar tract (lateral and medial motor columns of L1-L5 spinal cord segments) were counted blinded every 100 µm by the operator using a computer-assisted microscope (Olympus BX6 with Retiga 2000R camera) with the Stereoinvestigator software (MicroBrightField, Williston, VT, USA) at 40x magnification.

To investigate the activation of astrocytes and microglia cells in the lumbar tract of the spinal cord, immunohistochemistry for light microscopy was performed. The sections were incubated for 10 min in 3% H₂O₂ to quench endogenous peroxidase and preincubated for 1 h in 5% of NGS in PBS and 1% BSA. The slides were incubated overnight in anti-mouse GFAP or Iba1 antibodies to recognize astrocytes and microglia, respectively, (GFAP 1:500, Z0334 Dako; Iba1 1:500 019–19741 Wako) in 1% NGS in PBS. The sections were washed and incubated for 1 h in biotinylated goat anti-rabbit IgG (1:100, Vector Laboratories). The avidin-biotin peroxidase kit (ABC kit; Vector) and Novared kit (Vector) as signal revelation system was used. After mounting on slides, the sections were dehydrated through increasing grades of ethanol, cleared in xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany). For the analysis, cells were counted every 100 µm for a total of 30 sections for each animal. The astrocytes and microglial cells (GFAP or CD11b labeled cells) of the lumbar tract (L1-L5) were visualized and counted using a computer-assisted microscope (Olympus BX6 with Retiga 2000R camera) with the Stereoinvestigator software (MicroBrightField, Williston, VT, USA).