Nuance multispectral imaging system 3

Paraffin sections were deparaffinized and rehydrated, followed by antigen retrieval in Tris-EDTA buffer (pH 9) for 15 min in a microwave. Next, tissues were incubated with a cocktail of primary antibodies (Table 1). Subsequently, they were incubated with a cocktail of secondary antibodies, followed by incubation with a cocktail of tertiary antibodies tagged with fluorescence labels. Autofluorescence was blocked with Vector TrueVIEW autofluorescence quenching kit (Vector laboratories) for 5 min according to the manufacturer’s instructions. Afterwards, the tissues were incubated with DAPI for 10 min as counterstain and sealed. Image cubes were captured at a magnification of 20× using Nuance Multispectral Imaging System 3.0.1 (PerkinElmer) using NuanceFX 3.0.1 software (PerkinElmer). Filters used were 440:460 for DAPI, 490:530 for Alexa Fluor 488, 570:600 for Alexa Fluor 568, and 710:720 for Alexa Fluor 647. Spectral unmixing was performed with spectral libraries of each fluorophore subtracting the background signal.

Paraffin sections were deparaffinised and rehydrated, followed by antigen retrieval in Tris‐EDTA buffer (pH 9) in a 95°C water bath for 45 min. Tissues were incubated for 5 min with TrueBlack Lipofuschin autofluorescence blocker (Biotium, Fremont, CA, USA) in the dark at room temperature. Next, tissues were incubated with a cocktail of primary antibodies (Supplementary table 2). Subsequently, they were incubated with a cocktail of secondary antibodies, followed by incubation with a cocktail of tertiary antibodies tagged with fluorescence labels. Afterwards, the tissues were incubated with DAPI for 5 min as counterstain and sealed. Image cubes were captured at a magnification of 20× using Nuance Multispectral Imaging System 3.0.1 (PerkinElmer, Waltham, MA, USA) using NuanceFX 3.0.1 software (PerkinElmer). All filters available in the system were utilised to acquire the image cube with multiple wavelength acquisition (440:460 for DAPI, 490:530 for Alexa 488, 570:600 for Alexa 568, 710:720 for Alexa 647). Spectral unmixing was performed with spectral libraries of each fluorophore assigned different colours (DAPI = blue, Alexa 488 = green, Alexa 568 = red, Alexa 647 = yellow), subtracting the background autofluorescence. Colocalisation of the three fluorophores was analysed and assigned the colour cyan.