Rabbit anti p p65 ser536 antibody

After postfixation and incubation in gradient sucrose solutions, the L_3–5 segments of the spinal cord were sectioned into 30-μm frozen slices according to the method described above. The frozen slices were washed in PBS, and then high-pressure epitope retrieval (2 min) was performed in sodium citrate solution. The sections were incubated for 30 min in PBS containing 1% donkey serum and 0.5% Triton X-100 at 37°C and then incubated overnight at 4°C with a rabbit anti-p-p65 (Ser536) antibody (1:500, Cell Signaling Technology), a mouse anti-GFAP antibody (1:500, Cell Signaling Technology) a mouse anti-NeuN antibody (1:500, Abcam), or a goat anti-IBA-1 antibody (1:500, Abcam). The sections were rinsed in PBS three times for 15 min and then incubated for 30 min at 37°C with corresponding secondary antibodies (conjugated to Alexa FluorVR 488 and 594, Abcam). The samples were finally examined with a Fluorescence Inversion Microscope System (Leica, German), and images were acquired and processed using Leica software.

Proteins from liver tissues were isolated with RIPA buffer (Beyotime, Haimen, China) and
were quantified with a bicinchoninic acid (BCA) protein quantitation kit (Beyotime). Equal
amounts of proteins were separated by 5% or 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene
difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat
milk, the membranes were incubated with primary antibodies (rabbit anti-CYP2E1 antibody,
rabbit anti-CYP1A2 antibody, rabbit anti-p65 antibody, each 1: 1,000 diluted, purchased
from proteintech, Wuhan, China; rabbit anti-p-p65^Ser536 antibody, rabbit
anti-p-IkB^Ser32 antibody, rabbit anti-IkB antibody, each 1: 1,000 diluted,
purchased from Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After that,
the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit
secondary antibody (1:5,000 dilution, Beyotime) at 37°C for 45 min. Finally, the specific
protein bands were visualized with an enhanced chemiluminescent (ECL) reagent
(Beyotime).