Multiplex immunofluorescence analysis of CD3, CD79a, PD-1 and LAG-3
Discovery Red610 Kit
Multiplex immunofluorescence analysis of CD3, CD79a, PD-1 and LAG-3 was carried out on 4-μm FFPE sections using the Ventana Discovery Ultra platform 5-plex immunofluorescence protocol menu, which includes preset incubation temperatures and incubation times for antibody denaturation and multimer blocking. After deparaffinization (DISCOVERY Wash; Ventana, 950-510) and antigen retrieval (DISCOVERY CC1; Ventana, 950-500), the slides were blocked for 8 min (DISCOVERY Inhibitor; Ventana, 760-4840) and incubated with PD-1 (NAT105; Ventana, 760-4895) for 16 min. Sections were then incubated with Goat Ig Block as a multimer block, followed by OmniMap anti-mouse HRP (Ventana, 760-4310) for 12 min and chromogenic fluorescent detection using Discovery DCC Kit (Ventana, 760-240). After antibody denaturation and incubation with Goat Ig Block, the slides were incubated for 8 min with CD79a (SP18; Ventana, 790-4432). A subsequent incubation was performed with Goat Ig Block as a multimer block, followed by OmniMap anti-rabbit HRP (Ventana, 760-4311) for 12 min and chromogenic fluorescent detection using Discovery Red610 Kit (Ventana, 760-245). Before incubation for 8 min with CD3 (2GV6) (Ventana, 790-4341), the sections underwent a second antibody denaturation and blocking. Slides were then incubated with Goat Ig Block as a multimer block, followed by OmniMap anti-rabbit HRP (12 min) and chromogenic fluorescent detection using Discovery Cy5 Kit (Ventana, 760-238) before undergoing a third antibody denaturation and blocking and subsequent incubation with LAG-3 (EPR4392(2), 0.47-µg ml