Sc 52746

TMTH was supplied by Gyeongnam Agricultural Research and Extension Services, Jinju, South Korea. DNCB was procured from Sigma-Aldrich (St. Louis, MO, USA) and olive oil was obtained from a supermarket (South Korea). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-365062), TRAF6 (sc-8409), TNF-α (sc-52746), cyclooxygenase-2 (COX2; sc-376861) and inducible nitric oxide synthase (iNOS; sc-7271) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against MyD88 (4283S), IRAK4 (4363T), p-ERK (4377S), ERK (4695S), p-JNK (4668S), JNK (9258S), p-p38 (9215S) and p-38 (9212S) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Another serial section was pretreated with trypsin (Sigma-Aldrich) and 2 N hydrochloric acid for an antigen retrieval. The endogenous peroxidase was removed by 0.3% hydrogen peroxide, and the non-specific binding protein was treated with normal horse serum for 1 h. Then, the sections were incubated with primary antibodies at 4 °C overnight. The antibodies for COX-2 (160126, Cayman; 1:200), PARP (9545, Cell Signaling Technology Inc., Danvers, MA, USA; 1:100), TNF-α (sc-52746, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200) and BrdU (ab1893, Abcam, Cambridge, UK; 1:100) were used. Next day, the sections were incubated with a biotinylated secondary horse anti-mouse/rabbit IgG antibody and Vectastain Elite ABC reagents (Vector Laboratories Inc., Burlingame, CA, USA) for 1 h each. The immunoreactivity was visualized by a peroxidase substrate kit (Vector Laboratories Inc.). All sections were incubated in a humidity chamber, and rinsed with 0.01 M phosphate-buffered saline three times between each step. Cells occupying immunoreactive regions over 20% were regarded as positive, and the number was assessed in 10 regions of interest in each section by a histopathologist blinded to the groups.

Immunoblotting was performed as previously outlined by our laboratory [43 (link)]. Briefly, tissues were homogenized in protein lysis buffer (RIPA, Fisher Scientific, Pittsburg, PA, USA). 20 µg of protein lysates were separated on Mini-PROTEAN^® TGX™ Precast gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to nitrocellulose membranes. Membranes were blocked and incubated with polyclonal antibodies against Cldn6 (at a dilution of 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TNF-α (sc-52746, Santa Cruz Biotechnology, Dallas, TX, USA, 1:200) or IL-1β (ab9722, Abcam, Cambridge, MA, USA; dilution 1:200). Secondary (Ig)-horeseradish peroxidase antibodies were added for one hour at room temperature. The membranes were incubated with chemiluminescent substrate (Pierce, Rockford, IL, USA) for 5 min and the emission of light was digitally recorded using a C-DiGit^® Blot Scanner (LI-COR, Inc., Lincoln, NE, USA). Immunoblotting was conducted at least twice in triplicate and average band densities were normalized to β-actin densities prior to performing statistical tests.

Total cell extracts were prepared in 1× sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. Cell fractions were extracted with nuclear and cytoplasm protein extraction kit (Wanleibio, China). Cell proteins were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with GAPDH (1:1000, BM3876, Bosterbio, USA), p-PERK, p-IRE1, ATF4, P16 (1:500, bs-3330R, bs-16698R, bs-1531R, bs-20656R, Bioss, China), H3, NRF2 (1:500, D153567, D121053, Sangon Biotech, China), and TNF-α, IL-6, P21, MT1, MT2, HRD1, VCP, P65, p-P65, IKK (1:200, sc-52746, sc-32296, sc-136020, sc-13180, sc-13177, sc-293484, sc-136273, sc-514451, sc-166748, sc-7606, Santa Cruz, USA). Secondary anti-rabbit, anti-mouse antibodies (1:1000, BM2004, BA1001, Bosterbio, USA) conjugated with horseradish peroxidase were used. Detection was performed using a Thermo Scientific Pierce enhanced chemiluminescence western blotting substrate (Thermo Scientific). Results were analyzed by Tanon-410 automatic gel imaging system (Shanghai Tianneng Corporation, China).

The other serial sections underwent pretreatment with trypsin (Sigma-Aldrich) and 2 N hydrochloric acid for antigen retrieval. Endogenous peroxidase activity was removed using 0.3% hydrogen peroxide and a non-specific binding protein was blocked with normal horse serum for 1 h. Then, the sections were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal antibodies for TNFα (#sc-52746, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200) and BrdU (#ab8152, Abcam, Cambridge, UK; 1:100), and rabbit polyclonal antibodies for COX-2 (#160126, Cayman; 1:200) and cleaved PARP (#9545, Cell Signaling Technology Inc., Danvers, MA, USA; 1:100). The next day, the sections were incubated with horse biotinylated secondary anti-mouse IgG or anti-rabbit IgG antibody for 1h, followed by ABC reagents (#PK-6200, Vector Lab., Burlingame, CA, USA) for 30 min. Immunoreactivity was visualized using a peroxidase substrate kit (Vector Lab.) and counterstained with hematoxylin. The staining process was conducted in a humidity chamber, and sections were rinsed with PBS three times between each step. Immunostains omitting primary antibodies were used as the negative controls. Cells occupying immunoreactive regions over 20% were counted by a histopathologist blinded to the experimental groups.

The paraffin blocks of fixed lung tissue were sectioned (4 mm) onto microscope slides and incubated at 60 °C for 30 min. The slides were then immersed in xylene twice, and in 100% and 70% ethanol. After removing all remaining paraffin, the slides were rinsed with tap water and distilled water. To enhance the staining intensity, the washed slides were heated in pH 6 citrate buffer at 118 °C for 20 min. The slides were blocked with peroxidase blocking buffer for 10 min, and then immunostained with antibodies specific for anti-mouse Ly6G (Bio X Cell, BP0075-1) and TNF-α (Santa Cruz, sc-52746) O/N. After washing, Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies, A-11029) and Alexa Fluor 594-conjugated anti-rat IgG (Life Technologies, A-11007) were used as secondary antibodies and bound for 2 h. To suppress auto-fluorescence, Sudan Black B treatment was performed, and the slides were mounted with DAPI (Sigma-Aldrich, D9542) for nuclear counterstaining. Confocal images of neutrophils and TNF-α visualization were taken with a Leica TCS SP8 confocal system and processed with Leica Application Suite X software. The number of anti-Ly6G-positive neutrophils was counted manually from six random fields of confocal images and the integrated intensity of TNF-α fluorescence from multiple confocal images of each group was measured using Metamorph NX 2.0 software.