Immunostaining in Paraffin-Embedded Tissues

Tissues in paraffin block were sectioned at 4μm thickness and were processed for immunostaining. Sections were incubated with primary antibodies diluted (1:100) in 2.5% normal horse serum (NHS; Vector Labs) overnight at 4°C, then stained with biotin-conjugated secondary antibodies followed by incubation with peroxidase-streptavidin complex using Vectastain^® Universal Quick Kit (Vector Labs). Immunostaining was performed using 3, 3’-diaminobenzidine (DAB; Sigma) according to the manufacturer’s instructions. Sections were counterstained with haematoxylin for 1 min, mounted, and coverslip was permanently added for light microscopy. IHC scoring was done by using IHC profiler, an open source plugin, as described by Varghese et al. (2014) [17 (link)]. The score was assigned in a four tier system (0–3) viz. high positive (3+), positive (2+), low positive (1+) and negative (0).