Ta140003

IHC was used to investigate the expression of NGAL and FN. Rabbit monoclonal antibodies against NGAL (cat. no. ab216462) and FN (cat. no. ab268020) were purchased from Abcam. Paraffin-embedded sections were dewaxed by xylene, and endogenous peroxidase activity was quenched with 3% H₂O₂. Sections were digested with proteinase K for antigen retrieval. 2% BSA (cat. no. A2058; Merck KGaA) was used as the blocking reagent and incubated with the samples at room temperature for 30 min. Sections were incubated with primary antibodies (1:100) overnight at 4˚C and peroxidase conjugated goat anti-rabbit IgG (1:200, TA140003; OriGene Technologies, Inc.) at 4˚C for 30 min. IHC staining-positive areas were counted in 30 random cortex x400 high power fields (HPFs) under an Olympus microscope (IXplore; Olympus Corporation). Positive areas were measured using Image Pro 6.0 software (Media Cybernetics, Inc.) and expressed as percentage of positive areas per HPF.

Total protein was extracted from brain tissues using RIPA (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) containing PMSF (0.1 mM). The protein concentration was determined using BCA kit (Thermo Fisher Scientific, Inc.). The sample was mixed with the loading buffer and heated at 100˚C in a water bath for 10 min. Proteins (50 µg) were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (cat. no. ISEQ00010; EMD Millipore). Membranes were blocked using 5% skim milk at 4˚C for 2 h and washed with 0.1% TBST. Membranes were incubated with primary antibodies against phosphorylated (p) p38MAPK (ab31828; 1:1,000; Abcam), MAPK1 (ab31828; 1:5,000; Abcam), iNOS (ab213987; 1:5,000; Abcam) and GAPDH (ab22555; 1:2,000; Abcam) overnight at 4˚C. After three washes with TBST for 6 min, membranes were incubated with the secondary HRP-labeled goat anti-rabbit IgG antibody (TA140003; 1:5,000; OriGene Technologies, Inc.) at room temperature for 2 h. Membranes were washed three times with TBST for 6 min and placed into TBS. Enhanced chemiluminescence reagent (BB-3501; Cytiva) was used to detect the signal on the membrane. Images were acquired on a Bio-Rad image analysis system (Bio-Rad Laboratories, Inc.). The data were analyzed via densitometry using ImageJ software V2.1.4.7 (National Institutes of Health) and normalized to expression of the internal control GAPDH.