Alexa 488 labeled goat anti rabbit igg

Enzymatic retrieval was performed by incubating the sections in proteinase K for 10 min at 25°C. The sections were rinsed with PBS, permeabilized with 0.3% Triton X-100 in PBS for 30 min, blocked using blocking buffer (PBS containing 5% normal serum and 0.3% Triton X-100) for 1 h, and incubated with primary antibodies (4°C, 12 h) and secondary antibodies (37°C, 2 h) in PBS containing 0.05% Tween 20. The primary antibodies used were as follows: GFAP (1:100, Abcam, Catalog #ab7260), JAK2 (1:100, Abcam, Catalog #ab108596), p-JAK2 (1:100, Abcam, Catalog #ab32101), STAT3 (1:100, Abcam, Catalog #ab68153), p-STAT3 (1:100, Abcam, Catalog #ab76315), and neuritin (1:100, Abcam, Catalog #64186). The secondary antibodies Alexa 488-labeled goat anti-rabbit IgG (1:500, Catalog#A0423) and Alexa 647-labeled goat anti-rabbit IgG (1:500, Catalog#A0468) were purchased from Beyotime (Shanghai, China). Nuclei were stained with DAPI (Beyotime). Finally, slides were washed five times in PBS and coverslips were mounted in 90% glycerol for microscopic analysis.

Brains were dehydrated in graded alcohol and embedded in paraffin. The brains were cut into 5 μm thick coronal sections. Slices were blocked with 5% goat serum for 2 h and incubated with rabbit polyclonal anti-COX2 antibody (#ab15191, 1:200, Abcam) and mouse monoclonal anti-NeuN antibody (#GTX30773, 1:200, GeneTex, USA) overnight at 4 °C. The sections were incubated with Alexa 488-labeled goat anti-rabbit IgG (#A0423, 1:200, Beyotime, China) and Cy3-labeled goat anti-mouse IgG (#A0521, 1:200, Beyotime) for 1 h at 37 °C. The nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI, Beyotime) for 5 min at room temperature. The cells were imaged by an Olympus fluorescence microscope.
Neurons were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 in PBS and blocked with 5% goat serum for 2 h. The remaining steps were consistent with the method mentioned above.