Tissue tek optimal cutting temperature medium

In recorded mice, the rostral and caudal portion of the brain, and adjacent hippocampal sections to recorded section, were fixed in 4% paraformaldehyde (PFA) for a minimum of 24 h. Additional littermate mice were perfused with 0.1 M PBS for 1 min followed by 4% PFA for 6 min and 24 h post‐fixation in PFA. All samples were then washed in 0.1 M PBS before incubation in 30% sucrose for 48 h. Samples were embedded in Tissue‐Tek optimal cutting temperature medium (VWR International), and rapidly frozen on dry ice. Brain tissue sections were cut along the coronal axis in series (10 μm thick, 10 slides per series) using a Microm cryostat (HM 505 E) and collected onto SuperFrost Plus slides (Thermo Fisher Scientific), except for connexin‐labeled tissue (Figure 3), which was cut at 30 μm thickness and collected as floating sections into a 96 well plate in 0.1 M PBS.

Mice were anesthetized with a lethal mixture of ketamine/xylazine (1.5 x surgical dose) then transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde. Spinal cords were removed, post-fixed in 4% PFA for 2 h then transferred to 0.1 M PBS overnight before being cryoprotected by incubation in 30% sucrose for 48 h. Images of representative spinal cords were captured on an iPhone 6 s (Sup. Fig. 5a). Spinal cords were blocked into 10 mm segments centered on the impact site with the dorsal columns facing up, then embedded in Tissue-Tek optimal cutting temperature medium (VWR International), and rapidly frozen on dry ice. Tissue sections were cut in series (10 μm thick, 10 slides per series) using a Microm cryostat (HM 505 E) and collected on SuperFrost Plus slides (Thermo Fisher Scientific). Contused thoracic spinal cord segments were cut along the coronal (rostral-caudal) axis. Crushed thoracolumbar spinal cord segments were cut along the horizontal (dorsal-ventral) axis. Slides were stored at −20 °C until immunostaining.

Liver tissue samples were dissected and rapidly frozen in Tissue-Tek optimal cutting temperature medium (VWR, QLD, Australia) in liquid nitrogen vapor and stored at −80°C. Sections (8 µm) were cut using a cryotome (Thermo Fisher Scientific), air-dried, and fixed in acetone at −20°C. Prior to staining, sections were rehydrated in PBS/1% BSA for 60 min. Sections were incubated for 60 min with a biotinylated or purified antibody, washed three times with PBS/BSA and labeled for 60 min with a secondary reagent. The primary antibodies and the second labeling reagents are described in Table 1. Sections were mounted with polyvinyl alcohol mounting media with DABCO (Sigma-Aldrich) to prevent fading. Anti-Leishmania antibodies (clone V121, MHOM/IL/67/Jericho II) were purified from rabbit serum (a generous gift from Emanuela Handman, WEHI, Australia) (11 (link)). Immunofluorescence images were visualized using UltraView Spinning disk confocal microscope with Velocity Software (Perkin Elmer, MA, USA). All the images were processed using ImageJ version 1.50i (ImageJ, USA).