M glas

Microtome sections of fruiting structures formed by C. parasitica ICMP 11668 and its monokaryotic progeny monokaryons were prepared as previously described for C. aegerita [18 (link)], with a few minor modifications: cross sections of 8 µm thickness were prepared using a Rotary Supercut RM 2065 Microtome (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany) with a cutting speed of 50%. Sections were attached to glass slides with n-propanol [22 ]. The prepared slides were placed on a 50 °C heating block until they tightly stuck to the glass, and they were then stained with toluidine blue O as described by Herzog et al. [18 (link)]. Stained sections were dried again and covered with M-GLAS (Merck KGaA, Darmstadt, Germany) and a cover slip. Microscopic examination and composite image assembly was carried out at 200× magnification using an Axio Imager M2 (Carl Zeiss GmbH, Jena, Germany) equipped with a filter set 02 (Carl Zeiss GmbH, item number 488002-9901-000, suitable, e.g., for nuclear state fluorescence microscopy as applied in the present manuscript) and ZEN Works software version 2.6.

Tumor slices were placed at room temperature, fixed in acetone for 10 min and washed twice in TBS with 0.1% Tween 20 (TBS/T). Endogenous peroxidases were blocked with 1% H₂O₂/methanol, and slices were rinsed in distilled water and TBS/T for 5 min. Tumor sections were blocked with 2% rabbit serum diluted in TBS for 20 min and were rinsed for 5 min in TBS/T. CD31 was labeled with the rat anti-CD31 antibody for 1 hour (#550274 at 1/500; BD Biosciences, Pont de Claix, France) and the rabbit anti-rat antibody for 1 hour (#7077 at 1/750; Cell signaling Technology, Danvers, MA, USA). The labeling was detected with DAB for 5 min (#K3467; Dako, San Antonio, TX, USA) and rinsed with water for 5 min. Cell nuclei were stained with Gill’s hematoxylin for 2–3 s and washed with water 5 for min. Samples were dehydrated with 2 baths of 100% ethanol and one with xylene before the addition of mounting medium (M-GLAS, #1.03973.001; Merck Millipore, Fontenay sous bois, France). Images were taken using the Olympus BX-41 microscope with a 10× objective.
The evaluation of blood vessel number and diameter by CD31-quantification was performed on four fields of view for each tumor using Metamorph software.