Xylene

FFPE tissue blocks (n = 6 chemo-responsive and n = 6 non-responsive tissues) were water-bath-mounted onto PEN membrane slides (MicroDissect, Herborn, Germany) at 4 µm, as previously described [20 (link),21 (link)]. Briefly, the slides were deparaffinized by heating at 60 °C for 1 h, followed by rehydration using xylene (Chem-Supply, Gillman, Australia) for 90 s and then rinsed for 60 s with 100% ethanol (Merck, Bayswater, Australia). Slides were hematoxylin-stained and scanned using a NanoZoomer (Hamamatsu, Japan) at 43 × resolution (0.23 µm/pixel), and the tissues were annotated by an experienced pathologist using NPD.view 2.6.13 (Hamamatsu, Beijing, China). Approximately 1 mm² per region of each tumor tissue was excised by laser capture microdissection (LCM) using a Leica microscope (Leica Microsystems, Wetzlar, Germany) into individual centrifuge tubes. Excised tissues were lysed using 2% SDS (GE Healthcare, Parramata, Australia) in 10 mM citric acid, pH 6 (citric acid monohydrate, Sigma-Aldrich, Japan), followed by antigen retrieval at 98 °C for 90 min. The total protein concentration of each sample was estimated using a NanoDrop 2000 (Thermo-Fisher Scientific, Waltham, MA, USA) at 280 nm.

Samples were fixed in 1% paraformaldehyde (Santa Cruz Biotechnology, Dallas, TX, USA) for 4 hr at room temperature, embedded in O.C.T. TM Compound (Tissue-Tek, Sakura, Leiden, Netherlands) and flash frozen in liquid nitrogen. Cryosections of 10 μm thickness were mounted onto glass slides and stained with Safranin O (Sigma-Aldrich) for 10 minutes, dipped in 95% and 100% EtOH, cleared three times for 1 minute each in Xylene (Chem-Supply, GILLMAN, SA, Australia) and then mounted in Pertex medium (Grale HDS, Ringwood, VIC, Australia). For fluorescence analysis, 10 μm thickness slices were washed 2 times in PBS, permeabilized for 10 minutes in PBS-0.25%TritonX-100 (PBT) and then nuclei were stained by incubation with 5 µg/mL DAPI (Thermo Fisher Scientific Inc.) for 10 min at room temperature. The sections were washed in PBS, mounted with Fluoromount-G (Southern Biotech, Birmingham, AL, USA) onto glass slides and imaged using an epifluorescent Olympus IX70 inverted microscope using a SPOT Diagnostic RT-Slider camera and SPOT Diagnostic software using the indicated objective lenses. Images were processed using Photoshop software (Adobe).

Glycerol-free PNGase F (P0705L, 75,000 NEB units) was purchased from New England Biolabs (Ipswich, MA, USA). 2,5-DHB matrix was purchased from Sigma-Aldrich (Steinheim, Germany) and Bruker Daltonics (Bremen, Germany). Formalin was from Sigma-Aldrich. Trifluoroacetic acid (TFA), ethanol, and NaCl were from Merck (Darmstadt, Germany). Nitrocellulose membranes (0.025 μm VSWP) for dialysis were purchased from Millipore (Cork, Ireland). Xylene was purchased from Chem-Supply (Gillman, South Australia). Indium tin oxide (ITO) slides were purchased from Bruker Daltonics, while PEN membrane slides were from MicroDissect (Herborn, Germany). GLY3 standards (see Table 1) were purchased from Prozyme (CA, USA). Unless otherwise stated, all H₂O used was ultrapure (i.e., ≥18.2 MΩ and ≤5 ppb TOC).
Table 1

N-glycan standard mixture (GLY3) used for calibration of MALDI-TOF/TOF MS instrument

N-glycan composition	[M]	[M+Na]+	Concentration	Company
Man5GlcNAc2	1234.4333	1257.4225	1 pmol/μL	Prozyme, CA, USA
Man3GlcNAc5	1519.5659	1542.5551
Man3Gal4GlcNAc6	2370.8565	2393.8457