Publication protocol
Five-micron sections were deparaffinized, processed through a graded series of alcohols and rehydrated in distilled water per standard protocols. Heat-induced antigen retrieval was performed in citrate buffer (10 mmol/l concentration, pH 6) for 10 min. Tissue sections were incubated with peroxidase block (Dako, Carpinteria, CA, USA) or dry milk-based solution for 5 min to minimize background reactivity. The Dako Envision Plus Kit (Dako North America, Carpinteria, CA, USA) was used to perform the polymer-horseradish peroxidase-based IHC using the following antibodies: SLURP1 (Clone 569317; R&D Systems, Minneapolis, MN, USA; 1:100 dilution), CRISP3 (clone 295203, R&D Systems; 1:100 dilution), FLG (SPM181, Abcam, Cambridge, MA, USA; 1:25 dilution), ALOX15 (11-K, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100 dilution) and TNFAIP6 (FL-277, Santa Cruz Biotechnology; 1:200 dilution). Sections of stomach, small intestine and colon were used as positive controls for ALOX15 and TNFAIP6 in which inflammatory cells were positive. For FLG, SLURP1 and CRISP3, sections of normal esophagus were used as positive control. Negative controls where achieved by replacing the primary antibody by normal serum. Results were scored using a semiquantitative system for extent and intensity of staining. For extent, points were assigned as follows: 0=negative; 1= up to 10% positive cells; 2= 10–50% positive cells; 3= >50% positive cells. For intensity, points were assigned as follows: 0=negative; 1=weakly positive; 2=moderately positive; 3=intensely positive. A total score of 3 or higher was considered positive and 2 or lower was considered negative. Based on the pattern of staining for each one of the markers, ALOX15 and TNFAIP6 were scored in the full epithelial thickness; FLG and SLURP1 were scored in the mid and superficial levels of the epithelium; CRISP3 was scored in the basal cell layer peripapillary cells and superficial squamous cells. Owing to the discontinuous nature of eosinophilic esophagitis, ‘patchy’ lesions were graded in areas of histologic changes consistent with eosinophilic esophagitis. AM and MBR independently scored each section. Concordance was high and discrepant cases were reviewed to reach consensus.
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