Ab13243

The paraffin sections, 5 mm thick, which were drawn 48 h after reperfusion, were first deparaffinized in xylene, rehydrated with various grades of ethanol, and pretreated with 10 μg/ml proteinase K for 30 min at 37°C. By incubating the sections in 10% bovine serum albumin, nonspecific binding of immunoglobulins was blocked for 20 min. Then the sections were incubated overnight at room temperature in with anti-Nrf2 (ab31163, Abcam) or anti-HO-1 (ab13243, Abcam), and with anti-NeuN antibody to mark to neurons (monoclonal clone A60, Chemicon). Sections were then incubated with Alexa Fluor s 488 anti-rabbit IgG or Alexa Fluor s 594 anti-mouse IgG secondary antibodies for 2 h. Then, the sections were incubated with DAPI for nuclear counterstaining. Finally, the sections were coverslipped. The stained sections were examined under a fluorescence microscope (Olympus, Tokyo, Japan).

Proteins were extracted from cardiac tissue with radio immunoprecipitation assay (RIPA) lysis buffer (P0013 B, Beyotime) and their concentrations were determined with a bicinchoninic acid (BCA) protein kit (P0010S, Beyotime). The protein samples were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with 5% skim milk powder in TBS + tween (TBST) for 1 h and probed with primary antibodies (GAPDH, ab181602, Abcam; MMP-2, ab37150, Abcam; VEGFA, ab46154, Abcam; Nrf2, ab92946, Abcam; HO-1, ab13243, Abcam; NQO1, ab80588, Abcam) overnight at 4°C and shaken and rinsed three times with TBST solution. Then, the blot was incubated with diluted secondary antibodies (horseradish enzyme-labeled goat antirabbit IgG (H + L), ZB-2301, Chinese fir golden bridge) for 2 h. After washing, enhanced chemiluminescence (ECL) luminescence agent (P0018AS, Biyuntian) was evenly instilled. Finally, the FliorchemHD2 imaging system was used for scanning, analyzing, and collecting images as well as measuring the gray values of each band through Image J.

Proteins from Beas-2B and NHBE cells were extracted using the RIPA lysis buffer (Beyotime). Equal amounts of protein (40 μg/lane) were loaded on 10% SDS-PAGE and then transferred to nitrocellulose filter membrane. The membrane was incubated with the following primary antibodies and then incubated with secondary antibodies. The primary antibodies used in this study were: HO-1 (ab13243, Abcam, Dilution: 1:2000); Nrf2 (ab137550, Abcam, Dilution: 1:1000); p-P65 (ab86299, Abcam, Dilution: 1:2000); P65 (ab16502, Abcam, Concentration: 1:5000); and β-actin (ab8227, Abcam, Dilution: 1:2000). The following secondary antibody was used in this study: goat anti-rabbit IgG H&L (HRP, ab205718, Abcam, Dilution: 1:5000). Chemidoc XRS (Bio-Rad, Hercules, CA, USA) was used to detect protein bands.

Western blot analysis was performed as described previously [27 (link)]. One day after the behavioral tests, the rats were anesthetized with 10% chloral hydrate (3.5 mL/100 g), and the brains were collected. Total protein was prepared from the hippocampus, and the BCA assay was used to analyze the concentration of proteins (BCA Protein Assay, Thermo, 23228). The protein sample was resolved on SDS-PAGE and then transferred to a PVDF membrane. The membranes were probed overnight with the following primary antibodies at 4 °C: Nrf2 (Abcam, ab137550; 1:1000), HO-1 (Abcam, ab13243; 1:1000), ERK (Abcam, ab17942; 1:1000), p-ERK (Abcam, ab50011; 1:1000), NLRP3 (Abcam, ab214185; 1:1000), caspase-1 (Abcam, ab179515; 1:1000), and IL-1β (Abcam, ab9722; 1:1000). Subsequently, the membranes were washed with TBST (3 times for 5 min each) and incubated with goat anti-rabbit IgG (1:2000; Abcam, ab205718) and goat anti-mouse IgG (1:2000; Abcam, ab205719), as appropriate, for 1 h at room temperature. The immunoreactive bands were developed by chemiluminescence using Chemi Doc XRS System (Bio-rad, USA), and the intensities were normalized to that of GAPDH (1:10000; Servicebio, GB11002) used as an internal standard and quantified using ImageJ software.

Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (CWBio, Beijing, China) in the presence of protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentrations of the cell lysates were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of total protein were loaded in each well of a 12% sodium dodecyl sulfate polyacrylamide gel and subjected to electrophoresis. The proteins were then transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk and incubated with primary antibodies against GAPDH (10494-1-AP, ProteinTech), Nrf2 (ab62352, Abcam), HO-1 (ab13243, Abcam) and NF-κB (ab16502, Abcam) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies; detection was performed using ECL Plus western blotting detection reagents.

Moreover, 3-μm-thick sections of fixed (PFA 4%) frozen kidneys were cut with Cryostat Leica AS-LMD. Heme oxygenase 1 (HO-1) expression was studied using rabbit anti-mouse HO-1 (Abcam, Ab13243) followed by a polymer anti-rabbit IgG-HRP (DAKO, K4003). Staining was revealed with DAB solution. Slides were scanned by Nanozoomer (Hamamatsu). Hematoxylin–Eosin, Perl’s Prussian blue, and PAS coloration were performed by routine procedures using sections of paraffin-embedded kidneys at days 1, 2, and 4. Coloration of slides was scanned by Slide Scanner Axio Scan (Zeiss).