2.9. Immunohistochemistry

For immunohistochemistry (IHC), gills and posterior intestines from ASB were harvested and fixed in 4% PFA overnight at room temperature. Following fixation, the tissues were gradually dehydrated and embedded in paraffin before being sectioned into 7 μm slices. The paraffin-embedded slices were deparaffinized and hydrated with Histoclear (Thermo-scientific) and ethanol. Antigen retrieval was performed by heating the slides in sodium citrate buffer (10mM sodium citrate, 0.05% Tween 20, pH 6.0) under high pressure for 10 min. The slides were then blocked in 3% skim milk for 1 h at room temperature, followed by incubation with mouse anti-ASB IgT (1:20 dilution) overnight at 4 °C. The following day, the slides were rinsed in PBS thrice and incubated with the FITC labelled rabbit anti-mouse IgG (Dako) at 1:50 dilution for 2 h at room temperature. The slides were then washed thrice in PBS before the nuclei were stained with propidium iodide (PI) for 10 min at room temperature. A final wash was performed with PBS before the slides were mounted with ProLong™ anti-fade mounting reagent (Invitrogen). Microscopy visualization and images were collected using Olympus FV3000 confocal microscope.