Oct compound

Samples for histological analysis were fixed using 4% PFA for 30 ​min and washed three times in DPBS. Samples were subsequently transferred to cryomoulds (Tissue-Tek, ProSciTech, Kirwan, QLD, Australia), incubated in 30% BSA overnight and embedded in OCT compound (Tissue-Tek). Cryo-embedding of samples was performed using 2-methylbutane and liquid nitrogen. Sections of 5 ​μm were prepared using a Leica CM3050 cryostat at −23 ​°C. Sections were defrosted for 30 ​min at room temperature before staining with haematoxylin & eosin (H&E), Alcian Blue, Picrosirius Red or Masson's trichrome.

Following assessment of the bilateral hindpaw PWTs at ∼1 h post-EMA300 or vehicle administration, rats were euthanized with an overdose of pentobarbitone (Lethabarb^®, Virbac, Milperra, NSW, Australia) that was administered i.p. at a dose of ∼300 mg kg^-1 (i.e., 1 mL kg^-1 of the stock solution prepared as 325 mg mL^-1) per rat (Smith et al., 2013b (link)). A group of age-matched sham-control rats (n = 12) was also euthanized in a similar manner. Following laminectomy, the ipsilateral lumbar (L4–L6) DRGs were removed from all rats. These tissues were then processed for ex vivo investigations using immunohistochemistry (IHC) as well as western blotting (WB). For IHC, rats were perfused with 4% paraformaldehyde (Sigma–Aldrich, Sydney, NSW, Australia) in ice-cold 1x PBS (pH 7.4; ∼150–200 mL per rat) prior to extraction of the lumbar DRGs. Serial cross-sections (8–10 μm thick) were obtained from DRGs embedded in Optimal Cutting Temperature (O.C.T) compound (ProSciTech, QLD, Australia). Superfrost Plus^® slides (Thermo Fisher Scientific, Scoresby, VIC, Australia) were used to collect the cryosections. The slides were stored at -20°C until further processing for IHC. For WB, the lumbar DRG tissues were collected without perfusion and were snap-frozen using a dry-ice/ethanol slurry followed by immediate transfer to a -80°C freezer until further experimentation.