Opal multiplex ihc detection kit

Formalin-fixed paraffin-embedded sections of mouse spleen were stained with antimouse B220/CD45R (BD Biosciences; catalog no. 550286), anti-clPARP (Abcam; ab32064), and anti-Ki67 (Abcam; ab15580) antibodies using Opal Multiplex IHC detection kit (NEL810001KT; Akoya Biosciences). The sections were counterstained with 4′,6-diamidino-2-phenylindole for 5 min and mounted with ProLong Diamond Antifade mountant (Invitrogen; P36970). Images were obtained using Vectra Polaris multicolor fluorescence scanner (Akoya Biosciences). Quantification was carried out by inForm image analysis software (Akoya Biosciences). Normality tests were carried out for the B220⁺Ki67⁺ and B220⁺clPARP⁺ data. Both datasets were normally distributed, and accordingly, statistical significance was determined by unpaired t test.

Human and NHP lung tissues were placed in optimal cutting temperature compound (OCT, KMA-0100-00A, CellPath, UK) and snap frozen in liquid nitrogen. 10 µm sections were cut on to Super Frost microscope slides (12312148, Thermo Fisher) using a cryostat. Sections were air dried overnight at room temperature and then fixed in -20°C acetone for 10 minutes. Sections were air dried for <20 minutes prior to x2 washes with PBS. Multiplex immunofluorescence was performed on lung sections using a fully automated Bond-Rx Multiplex IHC Stainer (Leica Biosystems) and OPAL Multiplex IHC Detection Kit and counterstained with DAPI (Akoya Biosciences) according to a previously published protocol (27 (link)). T_RM were stained with purified, cross-reactive CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD69 (FN50, Biolegend) and CD103 (2G5.1, Bio-Rad, UK) antibodies. B_RM were stained with purified, cross-reactive CD20 (2H7, Biolegend), CD27 (M-T271, Biolegend) and CD69 (FN50, Biolegend) antibodies. Human lung (n=5) and NHP lung (n=3) sections were stained simultaneously using the same protocol and antibody concentrations. For full details, see online supplement.