P jak2 tyr1007 1008

The brains were dissected and fixed in paraffin. Coronal cryostat sections (20 μm) were stained with HE and immunofluorescent dyes. Brain sections were fixed at 4°C for 24 hours in 4% paraformaldehyde in PBS (0.01 M, pH 7.4), dehydrated in a series of graded alcohol dehydrations, and fixed in paraffin. The tissues were sectioned (5 μm) with a Leica® RM1850 rotary microtome (Leica Microsystems, Germany). The paraffin sections were dried at 60°C, dewaxed, and subjected to antigen retrieval. The sections were exposed for 1 hour at 37°C to primary antibodies targeting the following proteins: LCN2 (Abcam, 1 : 200), GFAP (Proteintech, 1 : 200), p-JAK2 (Tyr1007/1008) (Abcam, 1 : 100), and p-STAT3 (Tyr705) (Cell Signaling Technology, 1 : 200). The sections were washed twice with ice-cold PBS and then saturated with fluorescent secondary antibodies (Cell Signaling Technology, 1 : 100) in the dark for 1 hour. DAPI (4′,6-diamidino-2-phenylindole) (1 : 1000) was added in the dark for 2 min. The DAPI was rinsed 3 times with PBS for 1 min. The staining procedure for the cells was the same as above. For HE staining, sections were stained with alum HE. Colour images were obtained using a 20x laser scanning confocal microscope (Olympus FV1200, Tokyo, Japan).

The protein from tissues containing ischemic penumbra were harvested at 24, 48 h after cerebral ischemia was extracted by RIPA buffer (Beyotime Biotechnology, Shanghai, China) and was mixed with protease and phosphatase inhibitor cocktails (MCE, NJ, United States). The protein concentration was determined using a protein assay solution (Bio-Rad). Identical quantities of protein were denatured using protein loading buffer, loaded onto 10% SDS–PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes by electroblotting. The PVDF membranes were blocked by 5% bovine serum albumin (BSA) in TBST buffer for 1 h and were incubated overnight at 4°C using the following antibodies: LCN2 (Abcam, 1:2000 dilution), GFAP (Proteintech, 1:2000), p-JAK2 (Tyr1007/1008) (Abcam, 1:1000), JAK2 (Abcam, 1:2000), p-STAT3 (Tyr705) (Cell Signaling Technology, 1:2000), STAT3 (Cell Signaling Technology, 1:2000), and β-actin (Sigma, 1:5000). Reactive bands were detected using ECL detection reagent (Thermo Fisher Scientific, MA, United States) following the manufacturer’s instructions. The protocols for cell culture experiments were the same as those described above.