4.3. Single-Color Immunofluorescence Staining
ZED1227-TG2
For the single-color immunofluorescence staining, CUB7402, A161, and A083 were used as primary antibodies to detect total TG2, gliadin peptides, and ZED1227-TG2, respectively. Optimal working dilutions were determined for each antibody (1:1000 for CUB7402, 1:25 for A161, and 1:1000 for A083). The immunofluorescence staining protocol included de-paraffinization, high-temperature antigen retrieval (Tris-EDTA buffer, pH 9.0, 121C for 2 min), blocking of endogenous peroxidase activity, incubation with primary antibodies for 1 h, followed by incubation with anti-mouse horseradish peroxidase (HRP) polymer (30 min), and 1:200 diluted CF594-tyramide or CF488-tyramide fluorochromes (Biotium, Fremont, CA, USA). Hematoxylin was used for counterstaining and DPX mounting medium for embedding (Merck Life Science, Darmstadt, Germany). The staining procedure was carried out using an automated staining platform (LabVision Autostainer, ThermoFisher, Waltham, MA, USA).