Apotome widefield microscope

Pancreata were fixed in Z-FIX (Anatech) and paraffin embedded. Five µm sections were deparaffinized and subjected to antigen retrieval using Citrate unmasking solution (Vector H3300). Primary antibodies used: guinea-pig anti-insulin (1:250, Dako A0564), rabbit anti-glucagon (1:250, Immunostar 20076). Secondary antibodies: anti-guinea pig Alexa Fluor 488 (1:500, ThermoFisher A11073), anti-rabbit Alexa Fluor 555 (1:500, ThermoFisher A31572) Images were captured with a Zeiss Apotome widefield microscope.

Before immunohistochemistry or immunofluorescence was performed, hematoxylin and eosin (H&E) stainings of esophageal biopsies were examined to confirm proper orientation and inclusion of all layers of the epithelium. H&E stainings and immunohistochemistry of distal esophageal biopsies using mouse anti-IL-33 antibody (Nessy-1) were performed by the Pathology Research Core at CCHMC. Images were obtained using an Apotome widefield microscope (Zeiss, Thornwood, NY). For immunofluorescence studies, slides with 4-µm sections of FFPE esophageal biopsies underwent deparaffinization (serial incubations with xylene, 100% ethanol, 95% ethanol, 70% ethanol, 50% ethanol), antigen retrieval using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0), blocked with 10% donkey serum/phosphate-buffered saline (PBS), and then incubated with primary antibody diluted in 10% donkey serum/PBS overnight at 4 °C in a humidified chamber. The next day, slides were washed with PBS, incubated with secondary antibodies diluted in 10% donkey serum/PBS for 1 h at room temperature (RT) in a humidified chamber, and then washed in the presence of DAPI (0.5 µg/mL). Finally, a cover slip was added with ProLong Gold mounting reagent (Molecular Probes). The next day, slides were imaged using a Nikon A1R inverted confocal microscope. Analysis was performed with the Nikon Elements program.