Vectra multispectral imaging system

Excised tissues were formalin fixed and paraffin-embedded for antibody staining with a panel of fluorescent markers (CD4, mCherry, and CD8). Embedded sections were deparaffinized and hydrated prior to antigen retrieval and placement in blocking solution. Next, primary antibodies were sequentially applied upon removal of blocking solution at the following dilutions and incubation times: CD4 – 1:500 for 15 min, mCherry – 1:500 for 10 min, and CD8 –1:250 for 15 min. Secondary antibodies were then added following each primary antibody incubation using rat and rabbit secondary antibodies. The following staining dyes were added after secondary antibody washes at 1:100 dilution for 10 min: CD4 – Opal-dye 520, mCherry – Opal-dye 570, CD8 – Opal dye 620. Finally, stained sections were incubated in DAPI for 5 min at room temperature for nuclear labeling and mounted on coverslips for imaging. Imaging was performed at 40× using a Vectra multispectral imaging system (Akoya Biosciences) and a spectral library was generated to separate spectral curves for each of the fluorophores. Resulting images were analyzed using Nuance and inForm software (Akoya Biosciences).