Glial fibrillary acidic protein (gfap)

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).

A total of 60 male SD rats (body weight 210–250 g; mean 230 g) were purchased.
The C6 glioma cells were purchased from the Chinese Academy of Sciences, Shanghai Institute of Cell Biology (Shanghai, China). Factor (F) VIII R, glial fibrillary acidic protein (GFAP), and S-100 protein antibody I were bought from Sigma Company.
The following instruments and systems were also used: Immunohistochemical detection kit (Boster company. China); rat stereotactic apparatus (Jiangwan II); 4.7 T BIOSPEC30 type magnetic resonance tomography (Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences); and DIOMED60 type semiconductor laser diode (DIOMED company). The advanced fourth-generation aluminum gallium arsenic diode laser had a wavelength range of 790–830 nm and adjustable power output of 0.5–60 W. ThermaCAM S65 type infrared thermal image thermometer and thermocouple thermometer, both manufactured by Raytek company, were used for temperature measurement.