Optimal cutting temperature embedding medium

Dissected organs were fixed in 4% paraformaldehyde for 4–5 h and incubated in 30% sucrose at 4 °C overnight. Fixed organs were frozen in optimal cutting temperature embedding medium (Sakura Finetek, Japan) and 5-μm sections prepared at − 20 °C using a cryotome (Leica, Germany). Organs for paraffin blocks were fixed in Mildform 10N (Wako, Japan) overnight, embedded in paraffin, and sectioned using a MicromHM-335 E microtome (Microm International GmbH, Walldorf, Germany). Hematoxylin and eosin (HE), Masson trichrome (MT), and Oil Red O (ORO) staining were performed per established protocols. For immunohistochemical analysis of Mac2 expression in liver macrophages, frozen sections were incubated with a 1:200 dilution of rat anti-mouse anti-Mac2 antibody (Cedarlane, Canada), then Alexa Fluor 488-conjugated chicken anti-rat IgG secondary antibody (1:500 dilution; Thermo Fisher Scientific, MA) for secondary fluorescence staining. Finally, sections were mounted with Fluoromount (CosmoBio, CA) containing 2 μg/mL Hoechst and observed using a BIOREVO BZ-9000 microscope (Keyence, Japan).

For cryo-sectioning and subsequent LMD we only included control samples (ramet A) and inoculated samples collected 3 days after inoculation (ramet A and B). Prior to cryo-sectioning, phloem cubes (5 × 5 × 5 mm) were cut from inoculated bark samples 5 and 10 mm above the inoculation site and embedded and frozen in Optimal Cutting Temperature embedding medium (Sakura Finetek USA, Inc., USA). Similar-sized phloem cubes were cut from the control samples and processed in the same way.
Transversal cryo-sections (20 µm thick) were cut from the upper part of each phloem cube using a cryo-microtome (Microm HM 560 MV, Microm International GmbH, Walldorf, Germany). Optimal sections were obtained at the temperature of −18 °C set for both specimen and knife. Ten sections, intended for LMD, were placed on nuclease and nucleic acid free PET-membrane frame slides (1.4 µm; Leica MicroDissect GmbH, Herborn, Germany) and stored immediately in falcon tubes on dry ice. From each phloem cube we prepared two slides with five cross-sections per slide. The slides were stored at −80 °C for a few days prior to laser micro-dissection. Additional cryo-sections for morphological characterization were cut and processed as described below for tissue examined by light microscopy.

Archival formalin-fixed paraffin-embedded skin specimens were collected from five patients with in-transit advanced melanoma without irAEs, and from two patients with in-transit advanced melanoma with irAEs (Grades 2 and 3 hypophysitis), who were treated in the Department of Dermatology at Tohoku University Graduate School of Medicine. All patients gave written informed consent. The in-transit melanoma samples were processed for single staining of CD163a and POSTN, and the signal was developed with 3-amino-9-ethylcarbazole (Nichirei Bioscience, Tokyo, Japan). For cryosections, five cases of in-transit melanoma samples were frozen in optimal cutting temperature embedding medium (Sakura Finetek Japan Co. Ltd., Tokyo, Japan), and 6 μm thick sections were fixed in cold acetone for 30 min and blocked in immunofluorescence buffer (phosphate-buffered saline, 5% bovine serum albumin). Thereafter, each section was incubated with the relevant antibodies. The slides were mounted in DAPI Fluoromount-G (Southern Biotech, Birmingham, AL, USA) and examined using a Zeiss LSM 700 microscope equipped with a Spot digital camera.