Vectra 3 imaging system

Cryosections were fixed in formalin for 1 h at room temperature and dehydrated in an ascending ethanol series. A multiplex was stained by blocking endogenous peroxidase with hydrogen peroxide prior to incubation with anti-iba1 (polyclonal Ab #019-19741, Wako, Fijufilm, Japan), followed by incubation with the EnVision + polymer (Agilent, USA) and the OPAL system according to manufacturer’s instructions (Akoya Biosciences, USA). This staining cycle was repeated with anti-F4/80 (clone D2S9R, Life Technologies, USA). The following OPAL dyes were used for visualization: OPAL-520 for iba1 and OPAL-620 for F4/80. A second multiplex was stained in a similar manner as described above with anti-GFAP (clone 2.2B10, Invitrogen, USA) and visualization with OPAL-540, followed by anti-iba1 and visualization with OPAL-520. Nuclei were stained using 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Sigma-Aldrich, USA), and slides were coverslipped in Fluoromount G (Southern Biotech). Multispectral images were acquired using a Vectra^® 3 imaging system (Akoya Biosciences, USA). The inForm software (version 2.4.8) was used for spectral unmixing and cell segmentation as well as cell phenotyping. The cell phenotypes were quantified using R and RStudio (version 1.3.1073).

FFPE tissue sections (whole sections) were dewaxed and subjected to a heat-induced epitope retrieval step. Endogenous peroxidase was blocked by hydrogen peroxide prior to incubation with a monoclonal antibody against human CRMP1 (EP14521, Abcam, Cambridge, UK), followed by incubation with EnVision+ HRP-labeled polymer (Agilent Technologies Inc., Santa Clara, CA, USA) and visualization using the OPAL system (Akoya Biosciences Inc., Marlborough, MA, USA) according to manufacturer’s instructions. After protein inactivation, sections were incubated with a polyclonal antibody against human AHNAK (PA5-53890, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), followed by incubation with the EnVision+ polymer (Agilent Technologies Inc.) and visualization using the OPAL system. Nuclei were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Merck KGaA, Darmstadt, Germany) and slides were mounted in Fluoromount G (Southern Biotech, Birmingham, AL, USA). Multispectral images were acquired using a Vectra^® 3 imaging system (Akoya Biosciences Inc., Malborough, MA, USA).