Axiocam 105 colour

Slices (10 μm) were stained overnight with 1% w/v Luxol Fast Blue in 95% isopropanol:5% acetic acid (10%) at 60°C in a humidified water bath. Slices were washed briefly in 95% isopropanol followed by distilled water. Slices were differentiated in 0.5% w/v lithium carbonate for 30 s, followed by 70% isopropanol for 30 s followed by distilled water, then counterstained with cresyl violet. Slices (Bregma –1.94 mm) were imaged with a digital camera (Zeiss Axiocam 105 colour; Zeiss) attached to a stereomicroscope (Zeiss Stemi 305, Zeiss). The area of the contralateral corpus callosum was measured by a blinded investigator from the midline to a point directly above the distal end of the dentate gyrus (DG) granule cell layer²⁹ (link) using the area measurement function in ImageJ (FIJI).³⁰ (link)

Slices (10 μm) were stained with cresyl violet (Merck Millipore, Darmstadt, Germany) as described.¹⁶ (link) Slices were imaged with a digital camera (Scopetek DCM510; Scopetek Opto-Eletric Co. [Hangzhou, China] or Zeiss Axiocam 105 colour; Zeiss [Jena, Germany]) attached to a stereomicroscope (Wild model M8, Heerbrugg, Switzerland or Zeiss Stemi 305, Zeiss). The contusion was evident from a clear difference in the intensity of the cresyl staining. The area of the contusion was measured using image analysis software (Scopephoto 3.1, Scopetek Opto-Eletric Co. or Zen, Zeiss) by an investigator blinded to the experimental groups. Contusion volume was calculated by multiplying contusion areas, A, by the distance between brain sections, d (500 μm), according to the following formula: $\frac{d}{2}\ast \left(A_1+A_n\right)+d*\left(A_2+A_3+\dots +A_{n-1}\right)$
Secondary injury volume at 24 h was calculated by subtracting the mean primary injury contusion volume at 15 min from the total contusion volume measured at 24 h.

Somatic chromosome numbers (2n) were counted in fresh roots or very young flower bud meristems obtained from wild plants or rooting shoots of D. arbuscula, diploid and triploid cytotypes of D. cneorum subsp. cneorum, and D. cneorum subsp. arbusculoides. Individuals with counted chromosome numbers were simultaneously analysed for AGS and RGS and served as references for estimating ploidy in the remaining cytotypes and taxa. Freshly collected meristems were pre-treated in a 0.002 M water solution of 8-hydroxyquinoline for 3–5 h at low temperature (4 °C), washed in distilled water, fixed in a 3:1 mixture of 96 % ethanol and 98 % acetic acid for 24 h, and stored in 75 % ethanol. Samples were washed for 10 min in distilled water before and after being macerated in a 1:1 mixture of 35 % HCl and 96 % ethanol for 3 min prior to analyses. Microscopic slides and squashes were prepared using the cellophane square technique (Murín 1960 (link)). The slides were stained with a 7 % Giemsa stain solution, dried, and observed microscopically in a drop of immersion oil.
All chromosome spreads were analysed under 1000-fold magnification using a light microscope, and micrographs were taken using a ZEISS Axiocam 105 colour (Carl Zeiss, Vienna, Austria) and AxioVision LE64 v. 4.9.1.0 (Carl Zeiss, Microscopy GmbH).