Immunofluorescence staining and double immunostaining were performed as
previously described.28 (link) Briefly, four rats from each group were selected, deeply anesthetized,
and exsanguinated. Then, the lumbar enlargements were removed, soaked in 4%
phosphate-buffered paraformaldehyde for 4 to 6 h at 4 °C, and subsequently
dehydrated in a 10% to 30% gradient of sucrose in sterile water for five to
seven days at 4 °C. Next, the lumbar enlargement of the spinal cord tissues was
frozen in optimal cutting temperature (OCT) compound (Sakura, America) in a
cryostat at −25°C and then sliced at a thickness of 20 µm. The sections were
first blocked with 4% normal donkey serum, 0.03% Triton X-100, and PBS for 1 h
at room temperature. The sections were then incubated overnight at 4 °C with the
following primary antibodies: p-eIF2α (1:200, rabbit; Affinity), cleaved
caspase-3 (1:150, rabbit, Affinity), glial fibrillary acidic protein (1:1000,
mouse; Sigma), IBA-1 (1:250, mouse; Abcam), and NeuN (Mouse,1:1000, Abcam). The
sections were washed with PBST and incubated for 2 h with fluorescein
isothiocyanate- or Cy3-conjugated secondary antibodies (1:500, Abcam) at room
temperature. Finally, the stained sections were surveyed with an Olympus
fluorescence microscope, and images were acquired with a CCD Spot camera.
Finally, the images were analyzed using Image Pro-Plus 6.0 (Image Pro-Plus
Kodak, USA).
previously described.28 (link) Briefly, four rats from each group were selected, deeply anesthetized,
and exsanguinated. Then, the lumbar enlargements were removed, soaked in 4%
phosphate-buffered paraformaldehyde for 4 to 6 h at 4 °C, and subsequently
dehydrated in a 10% to 30% gradient of sucrose in sterile water for five to
seven days at 4 °C. Next, the lumbar enlargement of the spinal cord tissues was
frozen in optimal cutting temperature (OCT) compound (Sakura, America) in a
cryostat at −25°C and then sliced at a thickness of 20 µm. The sections were
first blocked with 4% normal donkey serum, 0.03% Triton X-100, and PBS for 1 h
at room temperature. The sections were then incubated overnight at 4 °C with the
following primary antibodies: p-eIF2α (1:200, rabbit; Affinity), cleaved
caspase-3 (1:150, rabbit, Affinity), glial fibrillary acidic protein (1:1000,
mouse; Sigma), IBA-1 (1:250, mouse; Abcam), and NeuN (Mouse,1:1000, Abcam). The
sections were washed with PBST and incubated for 2 h with fluorescein
isothiocyanate- or Cy3-conjugated secondary antibodies (1:500, Abcam) at room
temperature. Finally, the stained sections were surveyed with an Olympus
fluorescence microscope, and images were acquired with a CCD Spot camera.
Finally, the images were analyzed using Image Pro-Plus 6.0 (Image Pro-Plus
Kodak, USA).