Mrh53bl10

Immunohistochemistry (IHC) was performed on formalin fixed, paraffin embedded left colon biopsy tissue from healthy and ulcerative colitis patients. The sections were deparaffinized and subjected to 0.01 M citrate buffer (pH 6.0) antigen retrieval at 121 degrees for 4 mins in a decloaking chamber. Endogenous peroxidases were quenched by a 5 min incubation of 10% H₂O₂ in methanol. Non-specific binding was blocked by a 20 min incubation in Biocare background Sniper solution (BS966G, Biocare, Concord, CA21520, USA). Staining for human SLPI was performed using a specific antibody against human SLPI (PA5-20385, rabbit polyclonal, Thermo Fisher Scientific, Rockford, IL 61105, USA) at room temperature for 1 hour. Samples were then incubated with a horseradish peroxidase (HRP)-polymer (MRH53BL10, Biocare, Concord, CA21520, USA) for 30 mins, stained with Betazoid DAB chromogen (BDB900B, Biocare, Concord, CA21520, USA), counterstained with hematoxylin, dehydrated and mount in DPX.

Immunohistochemistry was performed on 10% (v/v) formalin fixed, paraffin embedded left colon biopsies. Sections were deparaffinized and subjected to 0.01 M citrate buffer (pH 6.0) antigen retrieval at 121 °C for 4 min in a decloaking chamber. Endogenous peroxidases were quenched by a 5-min incubation in 10% H₂O₂ in methanol. Non-specific binding was blocked by a 20-min incubation in BioCare background Sniper solution (BS966G, BioCare, Concord, CA21520, USA). Immunostaining was performed using specific antibodies against IL-1β (ab9722, Abcam, Cambridge, MA, USA) and NLRP3 (ab17267, Abcam, Cambridge, MA, USA) at room temperature for 1 h. An additional incubation with MACH 1 mouse probe (UP537L10, BioCare, Concord, CA21520, USA) for 15 min at room temperature was performed on sections incubated with the anti-NLRP3 antibody. Samples were then incubated with a horseradish peroxidase (HRP)-polymer (MRH53BL10, BioCare) for 30 min, stained with Betazoid DAB chromogen (BDB900B, BioCare, Concord, CA21520, USA), counterstained with hematoxylin, dehydrated and mounted with distyrene, plasticizer and xylene. Slides were examined using an IX71 microscope (Olympus Australia, Melbourne, Australia).