Cy3 goat anti rabbit igg h l

Ferrostatin-1 (Fer-1, HY-100579) and ML385 (HY-100523) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Lipopolysaccharide (LPS, L2880, 055:B5) and N-acetylcysteine(NAC, CAS#38520-57-9) were purchased from Sigma-Aldrich (St. Louis, MO, USA). FTH (DF6278, 1:1000) and xCT (DF12509, 1:1000) antibodies were purchased from Affinity Biosciences (Cincinnati, OH, USA). NOX4 (YN2975, 1:1000), PTGS2 (YT1073, 1:1000), NCOA4 (YT0302, 1:1000), NRF2 (YT3189, 1:1000), GPX4 (YN3047, 1:1000), β-Actin (YM3028, 1:5000), and HRP* Goat Anti Rabbit IgG (H + L) (RS0002, 1:10,000) antibodies were purchased from Immunoway (Newark, DE, USA). IL-6 (WL02841, 1:1000), TNF-α (WL01581, 1:1000), and Histone H3 (WL0984a, 1:500) antibodies were acquired from Wanleobio (Shenyang, China). HMGB1 antibody (T55060, 1:1000) was acquired from Abmart (Shanghai, China). Cy3 Goat Anti-Rabbit IgG (H + L) was purchased from Abclonal (Wuhan, China).

The histological observation was performed as in our earlier research [34 (link)]. In brief, the intestine was obtained from healthy tilapia as mentioned above. The collected intestines were fixed in Dietrich’s stationary liquid for 48 h and dehydrated by the gradient of ethanol (50%, 70%, 85%, 95%, and 100%) [34 (link)]. Afterward, samples were embedded in paraffin wax after vitrification by xylene. Intestinal sections with a thickness of 5 μm were dewaxed with xylene and then rehydrated by the gradient of ethanol (100%, 95%, 80%, 70%, and 50%). Samples were then treated with EDTA epitope repair solution (Beyotime, Shanghai, China) for 40 min at 98°C successively. Subsequently, samples were blocked using the QuickBlock™ Blocking Buffer for 30 min at 28 °C and rinsed twice. Afterward, samples were placed into the diluent of the anti-VIP antibody and anti-VIPR1 antibody and incubated for 1 h at 28 °C. Then, the former samples were rinsed five times and placed into a diluent of Cy3 goat anti-rabbit IgG (H+L) (AS007, ABclonal, Wuhan, China) for 1.5 h at 28 °C without light.
The former samples were then placed in 1 μg/mL DAPI (Beyotime, Shanghai, China) working solution for 5 min after washing five times with PBS. Finally, sections were washed with PBS, and imaging was done using the ZEISS Axioscope 5 microscope (Zeiss, Jena, Germany).

The reagents used were as follows: sodium new houttuyfonate (Yuanye, Shanghai, China, CAS: 112714-99-5); docetaxel (Yuanye, Shanghai, China); hydroxypropyl-β-cyclodextrin (HP-β-CD; Solarbio, Beijing, China); N-acetyl-cysteine (NAC; Macklin, Shanghai, China); matrix adhesive (Biozellen, Frontier, NE, USA); Opti-MEM I medium (Gibco, Billings, MA, USA); and crystal violet (BioSharp, Hefei, China).
The kits used were as follows: Cell Counting Kit-8 (Hycezmbio, Wuhan, China), ROS Detection Kit (Hycezmbio, Wuhan, China), BCA Protein Quantification Kit (Hycezmbio, Wuhan, China), Apoptosis Detection Kit (Hycezmbio, Wuhan, China); and Transwell chamber (Corning, NY, USA).
According to the protocol recommended by the manufacturer, the following antibodies were used for Western blot or immunofluorescence: Anti-BAX (Wanleibio, WL01637), Anti-p-GSK3β (Wanleibio, WL03683), Anti-β-actin (ABclonal, AC038), Anti-Bcl-2 (ABclonal, A19693), Anti-cleaved PARP p25 (ABclonal, A19612), Anti-caspase-9 (ABclonal, A0281), Anti-PDK1 (ABclonal, A0834), Anti-p-PDK1 (ABclonal, AP0426), Anti-AKT (ABclonal, A20799), Anti-p-AKT (ABclonal, WLP001), Anti-GSK3β (ABclonal, A11731), Anti-MMP1 (ABclonal, A22080), HRP Goat Anti-Rabbit IgG (ABclonal, AS014), Alexa Flour 594-Goat Anti-Rabbit IgG (ABbox, AD9279), and Cy3 Goat Anti-Rabbit IgG (H + L) (ABclonal, AS007).

Fixed samples were incubated with PBS containing 0.5% Triton X-100 for 20 min. PBS was added and washed three times, followed by blocking with 5% BSA for 30 min. Sections were then washed, and primary antibodies (AIM2, A3356, 1:100, Abclonal, CN; Caspase-1, 22915-1-AP, 1:100, Proteintch, CN; NLRC4, A7382, 1: 100, Abclonal, CN) were added and then incubated overnight at 4°C. A secondary antibody (Cy3 GoatAntiRabbit IgG (H+L) (AS007, 1:200, Abclonal, CN) or FITC Goat AntiRabbit IgG (H+L) (AS011, 1:200, Abclonal, CN)) was added and incubated for 50 min at room temperature (23°C ± 2°C), and cell nuclei were stained with DAPI.