Phosphate buffer saline egg albumin

We followed the methods of Méndez-Flores et al. [26 (link)]. Briefly, IL-22-expressing cells were determined in 4 μm thick sections of tissue. After deparaffinization and demasking of antigens, tissues were blocked with 3% H₂O₂. Then, nonspecific background staining was avoided with the IHC background blocker (Enzo Life Sciences). Tissues were incubated with goat polyclonal anti-human IL-22 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 10 μg/ml. Binding was identified with biotinylated donkey anti-goat IgG antibody (ABC Staining System; Santa Cruz Biotechnology). Slides were incubated with horseradish peroxidase- (HRP-) streptavidin, followed by incubation with the peroxidase substrate 3,3-diaminobenzidine (DAB) (Sigma-Aldrich) for 10 min. The sections were counterstained with hematoxylin. Negative control staining was performed with normal human serum diluted 1 : 100, instead of primary antibody, and the IHC universal negative control reagent (IHC universal negative control reagent, Enzo Life Sciences). The reactive blank was incubated with phosphate buffer saline-egg albumin (Sigma-Aldrich) instead of the primary antibody. Both controls excluded nonspecific staining or endogenous enzymatic activities [26 (link)]. Spleen and ganglion samples were used as a positive control ().

F4/80, UCP1 and TNF-α expressing cells were determined in 4 μm thick sections of available formalin-fixed paraffin embedded tissue. Endogenous peroxidase and binding of nonspecific proteins were blocked with 3% H₂O₂ and serum-free blocking solution (Enzo Life Sciences, Inc, Farmingdale, NY, USA), respectively. Tissues were incubated with biotinylated rat monoclonal antibodies (eBioscience, San Diego, CA, USA) diluted 1:500 for 18 h at 4 °C. Binding was identified with horseradish peroxidase-(HRP-)streptavidin (ABC Staining System; Santa Cruz Biotechnology). Slides were incubated with substrate 3,3′-diaminobenzidine (DAB) (SIGMA-Aldrich) for 10 min. The sections were counterstained with hematoxylin, dehydrated and mounted in resin. Negative control staining was performed with normal donkey serum diluted 1:100, instead of primary antibody. The reactive blank was incubated with phosphate buffer saline-egg albumin (SIGMA-Aldrich) instead of the primary antibody. Both controls excluded nonspecific staining or endogenous enzymatic activities.

Tissues placed on positively charged slides were incubated with mouse monoclonal anti-human IL-1, IL-6, IL-8, IL-10, IL-15, IL-22, IL-23, IFN-γ, TNF-α, and with rabbit polyclonal anti-human IL-2, IL-12p40, IL-17A, IL-21, or TGF-beta1 antibody (Abcam, Cambridge, MA, USA) or anti-human IL-4 antibody (Bio Legend Inc., San Diego, CA, USA) at 10 µg/mL during 30 min. Binding was detected with Universal Dako labelled streptavidin biotin reagent + peroxidase for primary antibodies from rabbit, mouse and goat (Dako, Glostrup, Denmark). Spleen and ganglion samples were used as a positive control. Negative controls were carried out with normal human serum (1:100) and with the immunohistochemistry universal negative control reagent (Enzo Life Sciences, Inc., Farmingdale, NY, USA), while phosphate buffer saline-egg albumin (SIGMA-Aldrich) was use in the reactive blank. Controls excluded nonspecific staining or endogenous enzymatic activities. We examined three different sections of each biopsy. As we have done before, cytokine-expressing cells were reported as the percentage of positive cells in three fields (X320) taken from the epithelium and lamina propria [27 (link)]. Results are expressed as the median, mean and 5th/95th percentiles.