Mouse monoclonal anti caspase 1 antibodies

The ileum segments embedded paraffin blocks were obtained from 13 neonatal infants (postnatal age range, 1–28 days) who needed surgeries, including 8 with NEC and 5 with intestinal atresia. Histomorphological examination was performed using haematoxylin and eosin (HE) staining; immunohistochemical (IHC) analysis were performed by BenchMark GX automated stainer (Roche). Rabbit polyclonal anti-NLRP3 (1:200, Abcam, UK) and mouse monoclonal anti-caspase-1 antibodies (1:50, Santa Cruz, USA) were used as primary antibodies. The horseradish peroxidase (HRP)-labelled secondary antibodies were used accordingly. Ventana UltraView Universal diaminobenzidine (DAB) detection kit (Ventana Medical Systems, Inc.) was used for visualization, and photomicrographs were obtained with an inverted microscope (Olympus, USA) at 100× or 400×.

Nuclear and cytoplasmic fractions were collected respectively using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China). Membrane proteins were extracted using the Membrane Protein Extraction Kit (Beyotime, China). Sample proteins were subjected to separation by SDS-PAGE, transferred to polyvinylidene difluoride membranes and blocked with 5% non-fat milk in TBST, followed by being blotted with primary antibodies and HRP-conjugated secondary antibodies accordingly. The primary antibodies used were: mouse monoclinic anti-TLR4 antibodies (Santa Cruz, USA), rabbit polyclonal anti-NLRP3 (Abcam, UK) and mouse monoclonal anti-caspase-1 antibodies (Santa Cruz, USA), rabbit polyclonal anti-NF-κB p65 antibodies (Abcam, UK), rabbit polyclonal anti-phospho-NF-κB p65 antibodies (Abcam, UK), mouse monoclonal anti- IκB-α antibodies (Santa Cruz, USA). Enhanced chemiluminescence (ECL) was to develop the signals. The images were captured using a ChemiDoc™ CRS + Molecular Imager (Bio-Rad, USA) and quantified by Image Lab software (Bio-Rad, USA).