Immunocruz abc kit

Lung tissues were formalin-fixed and embedded in paraffin. Five-micrometer sections were cut and dewaxed with xylene, hydrated, and the antigen retrieved in a citrate buffer (pH: 6.00, 98 °C) for 20 min. Endogenous peroxidase activity was blocked by 3% H₂O₂ for 10 min. Subsequently, the sections were incubated with 5% bovine serum albumin for 30 min. MPO heavy chain goat polyclonal (Santa Cruz, CA, USA) antibody was added and incubated overnight at 4 °C in a humid chamber. Afterwards, the sections were washed and incubated with biotin-labeled rabbit anti-goat secondary antibody. The sections were washed again and then incubated with an avidin‒peroxidase complex (ImmunoCruz ABC kit, Santa Cruz). Slides were stained with 3, 3′ Diamobenzidine (DAB, ChemCruz) to prompt the MPO to be visualized and then counterstained with hematoxylin to dye the cell nucleus. Dehydration with alcohol series was done and then sections were placed in xylene for differentiation. Finally, the sections were mounted using a DPX mount and visualized under a microscope, and image quantification was done using ImageJ software (Bethesda, Maryland, MD, USA).

Animal tissues were processed for histological analysis as previously described [20 (link)]. Briefly, tissues were fixed with 10% formaldehyde, embedded in paraffin blocks, and sectioned by the Augusta University histology core. The tissues were probed using antibodies to CgA (1:200; Immunostar, Hudson, WI, USA), Ki-67 (1:100; Dako Cytomation, Carpinteria, CA, USA), cleaved caspase-3 (1:500; Cell Signaling, Danvers, MA, USA), and 5-HT (1:5000; Immunostar, Hudson, WI, USA). Visualization of Ki-67 and cleaved caspase antibodies was done using the ImmunoCruz ABC kit (Santa Cruz Biotechnology, Dalls, TX, USA). To ensure the specificity of the 5-HT and CgA antibodies, we used a commercially available mixed IgG pool as an isotype control that stained nothing in our sections. Goblet cells were visualized using Alcian Blue Periodic acid schiff (AB/PAS) staining which was performed by the Augusta University histology core. Histological quantification and analysis was performed separately by two individuals blinded to treatments. At least 10 different sections containing approximately 8 crypts per section were counted for each mouse. The cGMP level in mouse colon mucosa was measured as previously described [23 (link)] using a cyclic GMP EIA kit (Cayman, Ann Arbor, MI).