Thionin acetate

The R series of tissue sections (adjacent to those that underwent immunohistochemical staining) was washed 5 × 5 in TBS and mounted onto gelatin-coated slides, dehydrated in ascending concentrations of ethanol (50%, 70%, 95%, 100%), and defatted with xylene. Sections were then rehydrated and stained with a 0.25% w/v thionine solution (sold as “thionin acetate” by Sigma-Aldrich, St. Louis, MO, USA; MW 287.34 g/mol; cat #T7029; ([97 ], p. 151); [98 (link)]). A 0.4% glacial acetic acid solution was used to remove excess thionine and the tissue slides were dehydrated in ascending concentrations of ethanol solutions (50%, 70%, 95%, 100% × 3), and then cleared in mixed-isomer xylenes before being coverslipped using DPX mountant for bright-field microscopic observation (Section 2.6.1).

Pairs of sections immediately adjacent to the sections that were sampled for TH staining were selected from a subgroup of the brains at PN14 and PN545 (n = 3 per group and n = 7–8 per group, respectively). These sections were stained with 0.1% filtered thionin-acetate (Sigma, St Louis, Missouri, USA, T7029), dehydrated through a series of alcohol rinses, mounted with DPX (dibutylphthalate polystyrene xylene), and coded. Images were taken at a magnification of ×220, printed, and montaged together. The identifying features from the TH sections were matched to the corresponding features on the thionin section to enable the borders to be drawn around the midbrain region of interest. Stereological analyses were then undertaken using Cavalieri’s method and the physical disector technique as described above. This yielded the absolute number of all neurons within the VTA, SNCd, SNCv, and RRF of PN14 repeated normoxia versus repeated hypoxia rats. At PN545, the absolute number of all neurons within the VTA, VTA + Cli, and SNCd of repeated normoxia versus repeated hypoxia rats was obtained.

Detached leaves treated with crude enzyme solution and B. cinerea spores were incubated for 3 dpi under the same conditions. Each leaf was fixed with FAA solution (5% formaldehyde, 70% ethanol, and 5% acetic acid) for 48 h. The fixed samples were dehydrated with ethanol (70% for 24 h and 85% for 3 h) and subsequently displaced with butanol through a butanol solution series (35% for 1 h, 55% for 1 h, 75% for 1 h, and 100% for 24 h). Samples were then embedded in Paraplast Plus (Sigma-Aldrich, St. Louis, MO). Tissue sections (15 μm thick) were prepared using a rotary microtome (Leitz, Austria). The samples were dewaxed in xylene, and treated sequentially with anhydrous ethanol and 70% ethanol. Then, the samples were stained according to Stoughton⁴⁴ with some modifications. First, staining was done with 0.025% (w/v) thionin acetate (Sigma-Aldrich) in 0.1 mM sodium acetate and a saturated solution of Orange G dye (Sigma-Aldrich), before rinsing with a solution of ethanol and xylene (1:1). Stained sections were mounted on glass slides and observed under a BX50 light microscope with an FX380 CCD camera system (Olympus, Tokyo, Japan).