Histology and Immunohistochemistry

Formalin fixed tissues were processed by routine histology method. Hematoxylin and eosin staining and immunohistochemistry (IHC) against PRCV nucleoprotein (N) were performed on serially sectioned formalin-fixed paraffin-embedded tissues. Briefly, 4 μm sections were dewaxed and dehydrated through xylene and graded alcohol, endogenous peroxidase activity quenched with a methanol and hydrogen peroxide, followed by unmasking of epitope with heat-mediated antigen retrieval in pH 9 buffer at 100°C for 10 mins (Dako). Slides were assembled into Shandon Sequenza cover plates to facilitate IHC (Shandon, USA). Tissue sections were incubated with an anti-N mouse monoclonal antibody DA3 at 0.1 µg/ml or concentration matched isotype mouse monoclonal antibody for 1 hr at RT and then with Dako ENVISION™ polymer for 20 min at RT. Immunolabelling was performed at room temperature and sections were washed three times with Tris-buffered saline between incubations. Immunolabelling was visualized using 3,3-diaminobenzidine (Sigma Aldrich) and counterstained in Mayer’s haematoxylin (Surgipath, UK). Slides were dehydrated in absolute alcohol, cleared in xylene and mounted using Dibutyl Phthalate Xylene and glass coverslips. A serial section was stained with haematoxylin and eosin for histopathology evaluation. Positive control slides containing either TGEV or PRCV infected or uninfected cells prepared in agarose matrix were included during IHC experiment to confirm immunolabelling (32).