Elemental red phosphorus

Solvents were purchased from Merck Millipore (Bayswater, VIC, Australia), Chemicals including 2,5-dihydroxy benzoic acid, elemental red phosphorus and Supra pure® nitric acid (70%) and hydrochloric acid (30%) were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Embedding and freezing supplies including cryofilm fitting tool set (2.0 cm), embedding container (1.5 cm × 2.0 cm), cryofilm fitting tool, embedding medium (SCEM), cryofilm 2C (9) (2.0 cm in width) were purchased from Section-Lab Co. Ltd. (Tokyo, Japan). Sectioning supplies including Menzel-Gläser Superfrost Ultra Plus Glass slides, optimal cutting temperature (OCT) compound and Feather® C35 tungsten microtome blades were purchased from Grale HDS (Ringwood, Australia). Lysing Matrix Tubes with 0.5 g Lysing Matrix D (1.4 mm ceramic spheres) were purchased from MP Biomedicals (Seven Hills, NSW, Australia). Elemental standards were purchased from PerkinElmer (Melbourne, VIC, Australia).

The lymph nodes were cryosectioned at 10 µm thickness and mounted onto indium tin oxide (ITO) slides (Bruker Daltonics, Billerica, USA). The slide was prepared for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging following a previously established method (35 (link)) to sublimate recrystallized 2, 5-dihydroxybenzoic acid (DHB) (Sigma) onto the ITO slide. The slide was then loaded into the UltrafleXtreme (Bruker) mass spectrometer. Elemental red phosphorus (Sigma) was used to calibrate the instrument. The sections were then ablated at 15 µm raster width (i.e. 15 µm spatial resolution) and full scan analyzed at m/z 380-1800. The peaks and images were analyzed on SCiLS lab (Bruker).