Vectra polaris

Multispectral analysis of FFPE tissue utilised the MOTiF™ PD-1/PD-L1 kit (Akoya Biosciences, cat# OP-000001). Staining with Leica BOND RX (Leica Biosystems) and imaging with Vectra^® Polaris™ (Akoya Biosciences) was performed at The Walter and Eliza Hall Institute (WEHI) Histology core facility as per kit manufacturer instructions. Briefly, tissue was stained through cycles of incubation with primary antibody, anti-IgG polymer HRP and covalent labelling with Opal TSA fluorophores, followed by heat induced epitope retrieval to remove bound antibodies prior to subsequent antibody cycles. Target markers included CD8 (Opal 480), PD-L1 (Opal 520), PD-1 (Opal 620), FoxP3 (Opal 570), CD68 (Opal 780, PanCK (Opal 690) and spectral DAPI DNA stain. Whole slide multispectral scanning was performed on the Vectra^® Polaris™ using automatically adjusted exposure settings. Image tiles were spectrally unmixed in InForm^® (Akoya Biosciences), then restitched in QuPath software (34 (link)).

Mice ear cryosections (6 µm) were fixed in acetone for 10 min at RT and air-dried. Then, cryosections were incubated with 400x diluted Alexa Fluor 488-conjugated rat-anti-mouse Ly-6G (GR-1 staining, eBioscience) for 1 hour at RT. After washing in PBS, nuclei were stained using DAPI (Invitrogen) at 1 µg/ml for 5 minutes at RT and embedded with fluorescence mounting medium. Tile scanning to obtain an image of the whole ear was performed using the Vectra^® Polaris™ (Akoya Biosciences) microscope with the following settings: DAPI MSI 0.43ms, FITC 81.70ms and a 20 times magnification. GR-1 staining of cryosections was analyzed with ImageJ/Fiji software. The total area (µm²) of mice ears and the area of the specific GR-1 staining (µm²) was measured. Quantification was calculated as the GR-1 area (µm²) divided by the total area (µm²). The following formula was used to determine the GR-1/total area ratio:

Muscle fiber types were assessed on both operated and contralateral MDN. After electrophysiological assessment, the entire MDN was carefully dissected and removed. Afterward, muscles were embedded in optimal cutting temperature compound (Tissue-Tek) using liquid-nitrogen-cooled isopentane and stored at −80°C for 24 h. Cross sections 10 µm thick of the embedded muscles were then obtained. A modified version of a previously described immunofluorescent protocol was used (Bergmeister et al., 2016 (link), 2019 (link)). First, Wheat germ agglutinin conjugated Alexa Fluor 594 (1:250) was applied for 10 min to stain the muscle fiber membrane. Primary antibodies diluted in 0.1 m PBS with 10% goat serum against myosis heavy chain (MHC)-I (BA-F8; 1:50), MHC-IIa (SC-71; 1:600), and MHC-IIb (BF-F3; 1:100) were used and applied for 60 min (Developmental Studies Hybridoma Bank). Afterward, the secondary antibodies against Alexa Fluor 633 immunoglobulin G2b (IgG2b; 1:250), Alexa Fluor 488 IgG1 (1:250), and Alexa Fluor 555 IgM (1:250) were applied for 60 min (Life Technologies). Entire cross sections of the muscle were acquired using a whole-slide scanner (Vectra Polaris, Akoya Biosciences) and subsequently analyzed using the Halo imaging analysis platform version 3.2.1851.421 (Indica Labs) by one independent investigator (M.L.).

On sequential TMA sections, each biomarker was stained by either chromogenic IHC for PD-L1, CK, CD68, or CD8, or the optimised mIF panel for PD-L1, CK, CD68, CD8, and DAPI. Optimised retrieval methods and staining conditions are detailed in Supplementary Table S1, and were as described by [4 (link),17 (link)]. CK was applied to serial Section 1; PD-L1 to serial Section 2; mIF panel for PD-L1, CK, CD68, CD8, and DAPI to serial Section 3; CD68 to serial Section 4; and CD8 to serial Section 5. DAB IHC slides were scanned at ×20 on an Aperio AT2 digital scanner (Leica Biosystems, Milton Keynes, UK) and mIF slides were scanned on an Akoya Vectra Polaris (Akoya Biosciences, Marlborough, MA, USA) at ×20 using the MOTiF™ protocol. Images were automatically stored on a secure networked server. Comparable quantitation of single channel mIF with the DAB IHC on sequential sections was undertaken by DIA.