Ab115819

Streptozotocin (STZ) (S0130) was procured from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) and Atripla, a fixed-dose combination antiretroviral drug (cART) was purchased from Bristol-Myers Squibb and Gilead Sciences (Foster City, CA, USA). The primary antibodies interleukin-1beta (IL-1β) (ab2105), interleukin-6 (IL-6) (ab9324), tumor necrosis factor-alpha (TNF-α) (ab6671), inducible nitric oxide synthase (iNOS) (ab115819), malondialdehyde (MDA) (ab243066), 8-hydroxydeoxyguanosine (8-OHDG) (ab62623), caspase 3 (ab4051), and Ki-67 (ab15580) were purchased from Abcam (Cambridge, MA, USA). The biotinylated goat anti-rabbit (BA-1000) and goat anti-mouse (BA-9200) secondary antibodies, and Avidin–Biotin Complex kit (ABC) (PK-6100) were purchased from Vector Laboratories (Burlingame, CA, USA).

Immunohistochemistry was conducted following the method in a previous report [23 (link)]. Antibodies including LC3B (1:600, #ab192890, Abcam, UK), p62 (1:100, #ab207305, Abcam, UK), Beclin1 (1:700, #ab207612, Abcam, UK), CD86 (1:400, #ab220188, Abcam, UK), iNOS (1:100, #ab115819, Abcam, UK), and CD163 (1:1000, #16646-1-AP, Pro-teintech, China) were used to detect protein expression in tissues. Images were captured at 100x and 400x magnification with the same parameters, and five fields of view per sample were randomly selected to assess the expression levels of the protein. Next, the percentage and the staining intensity of positive cells in each image were analyzed automatically by the “IHC profiler” plugin in Image J software (Version: 1.52a). H-score (H-score = (1 × (% of cells 1+) + 2 × (% of cells 2+) + 3 × (% of cells 3+)), 1 = lack or weak expression, 2 = moderate expression, 3 = strong expression) was applied to quantify the IHC images [24 (link),25 (link),26 (link)]. The average H-score from five fields of view was defined as the final score of the sample. Samples with poor immunohistochemical staining were removed.

Mouse colon tissues were embedded in paraffin and sliced with an ultra-thin microtome. Slices were then deparaffinized with xylene, rehydrated with gradient alcohol, and incubated with 3% H₂O₂ to block activity of endogenous peroxidase. Subsequently, the slices were boiled in 10 mM sodium citrate (pH 6.0) for 30 min, then blocked with 10% normal goat serum for 15 min, and incubated with the corresponding Abcam (Cambridge, UK)-purchased primary rabbit antibodies to NLRP3 (ab214185, 1: 100), arginase 1 (Arg1, ab233548, 1: 2000), iNOS (ab115819, 1: 100) overnight in a damp room at 4 °C. The following day, slices were incubated with the corresponding secondary antibodies goat anti-rabbit horseradish peroxidase (HRP)-labeled immunoglobulin G (IgG) (ab205718, 1: 2000) for 1 h at room temperature, and a 2,4-diaminobutyric acid (DAB) kit (Invitrogen, Carlsbad, CA, USA) was used for immunoreactivity detection.

Mouse gastric tissues were prepared for hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining as described previously [20 (link)]. H&E staining was conducted to evaluate the mucosal and inflammatory cell infiltration of the gastric cells. The histopathologic grades were classified based on the severity of inflammatory cell infiltration: level 0 (no inflammatory cells), level 1 (minimal), 2 (mild), 3 (moderate), 4 (marked), and 5 (severe), as described previously [25 (link)].IHC staining was performed by using antibodies against COX-2 (PA5-88606, Thermo Fisher Scientific, Waltham, MA, USA) and iNOS (ab115819, Abcam, Boston, MA, USA), respectively. The tissue sections were then incubated with ImmPRESSHRP Universal Antibody (MP-7500, Vector Laboratories, Newark, CA, USA), and finally developed with an ABC kit (ImmPACT DAB SK-4105, Vector Laboratories). The stained tissues were then analyzed using a microscope (AXIO IMAGER M2, Carl Zeiss, Oberkochen, Germany). The image was analyzed the intensity of protein expression using ImageJ (National Institute of Health, Bethesda, MD, USA), as previously described [26 (link)]. Five fields were randomly selected per sample to calculate the mean intensity and compared to the control group (100%).

The carotid sections were immersed in antigen retrieval solution and dried in an oven at 60°C for 30min. The sections were then put into 3% H₂O₂ for 10min to block the endogenous peroxidase activity and incubated with normal goat serum for 15min at room temperature. Subsequently, primary antibodies were added in the sections separately at 4°C overnight in a humid chamber (anti-α-smooth muscle actin (α-SMA), ab5694, Abcam, United States; anti-Macrophage/Monocyte (MOMA-2), GTX39773, GeneTex, United States; anti-iNOS, ab115819, Abcam, United States; anti-arginine 1 (Arg-1), ab23354, Abcam, United States). After that, the sections were incubated with Biotinylated Goat anti-Mouse or Goat anti-Rabbit IgG (1:200) for 2h. After development with 3,30-diaminobenzidine (DAB) and counterstaining with hematoxylin, a light microscope was used to photograph the sections. Average optical density of staining area was semi-quantitatively calculated by Image-Pro Plus 6.0 software.