M o m block

Sections (5 µM thick) were taken from paraffin embedded blocks. Slides were dewaxed and brought to dH₂O down an ethanol gradient. Masson’s trichrome staining, and immunoperoxidase staining for neutrophils and macrophages, was performed as described in Wilkinson et al. (2019a) (link). For immunofluorescent staining, antigen retrieval was achieved with citrate buffer and sections blocked in goat serum and M.O.M block (Vector Laboratories, CA, United States). Rabbit anti-keratin 14 (clone: Poly19053; Biolegend, CA, United States), mouse anti-TNF-α (clone: 52B83; Abcam, Cambridge, United Kingdom) and rat anti-CD107b (clone: M3/84; BD Biosciences, NJ, United States) primary antibodies were detected with Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific) and slides were mounted with MOWIOL 488 containing DAPI (Thermo Fisher Scientific). Slides were imaged on an LSM 710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) using a 20× objective lens and 405-nm diode, 488-nm argon, and 561-nm diode-pumped solid-state lasers. Staining was quantified using ImageJ v.1.8.0 (National Institutes of Health, MD, United States).

After whole brain extraction and fixation in 4% paraformaldehyde for 24 h followed by incubation in 30% sucrose in phosphate-buffered saline, 50 μm sections were used for examination with ultra-confocal microscopy. Lungs and livers were also obtained for microscopic examination. For immunofluorescence studies, 50 μm sections were rinsed twice in phosphate buffered saline, pH 7.4 (PBS), and placed in 10 mM sodium citrate buffer, pH 8.5, for 30 min at 80 °C for antigen retrieval. Following 3 brief washes in PBS, sections were then blocked and permeabilized in 10% normal donkey serum containing 0.3% Triton X-100 for 30 min and transferred to MOM block (Vector Labs) for 1 hr at room temperature (RT). After 3 additional washes in PBS, primary antibody cocktails were diluted in blocking solution and left overnight at RT. The following antibodies were used: antibodies against Serpin-B2 at 1:200 (PAI-2 Antibody: sc-6649 Santa Cruz Biotechnology) and Glut-1 at 1:500 (400060-50UG, EMD Millipore) according to the manufacturers’ instructions.