Axioscope

After collection, testicular samples, divided into 4–5 pieces from each group (control, low-, intermediate-, and high-level LDR radiation), were sectioned and immediately fixed in 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS, at 4 °C for at least 48 h before processing for LM and TEM as previously described [31 (link),32 (link)]. Semithin sections (1 µm thick) were stained with methylene blue and images were taken using a digital camera (Leica DFC230) mounted on a LM (Zeiss Axioscope, Carl Zeiss Microscopy GmbH, Jena, Germany). Ultrathin sections (60–80 nm) were cut with a diamond knife, mounted on copper grids, and contrasted with UranyLess and Lead citrate (SIC, Rome, Italy). Images were captured using Philips TEM CM100 electron microscopes operating at 80 kV [33 (link),34 (link),35 (link)].

For the analysis of early seed development, the oldest closed flower bud of a given inflorescence was emasculated. One day after emasculation, the flowers were pollinated with wild-type pollen and harvested 24 h later.
For whole-mount clearings flowers were vacuum infiltrated in an ethanol:acetic acid solution (9:1) for 30 min, kept at 4°C overnight, washed for 1 h each with 80% and 70% ethanol, and mounted in chloral hydrate:glycerol:water solution (8:2:1; w:v:v). Cytochemical staining of GUS activity was performed on samples as described previously (Vielle-Calzada et al., 2000 (link)). GUS-stained samples as well as cleared whole mounts were then visualized under a Zeiss Axioscope (Zeiss, Oberkochen, Germany) and images were captured by a Canon PowerShot G10 camera. Fluorescence signals were detected by a Leica DMI6000B microscope (Leica Microsystems, Wetzlar, Germany).

In order to determine the size of the villi and crypts in the ileum, as established indicators of intestinal health [45 (link),46 (link),47 (link)], the histological samples were processed as follows: During the dissection, a tissue sample of approximately 1 cm² was removed from the ileum (halfway between the diverticulum and the cranial part of the cecum) and fixed in 4% formaldehyde for 48 h. After fixation, tissue samples were embedded in paraffin using standard techniques [48 ]. For histological evaluation, 4 µm sections of all samples were stained with hematoxylin and eosin (HE) using established protocols [48 ]. The villus height was measured from the tip of the villi to the villus crypt junction; villus width was measured at the base of the villus above the villus crypt junction; the depth of the crypts of Lieberkuhn was measured from the villus crypt junction to the basal lamina of the crypts (just above the Lamina muscularis mucosae). The relation of the villus height to the depth of the crypt was computed at the end [49 (link)]. Measurements were performed using a Zeiss Axioscope (Carl Zeiss Jena GmbH, Jena, Germany).

Wound healing assays were used to access cell migratory activity with IBIDI Culture-Inserts (80,209, 35 mm, USA). The ATG4B silenced cells (2 × 10⁵ cells) were seeded into an insert for 16 h and removed for cell migration. The healing distances were quantified and measured with image J and tabulated with Prism 5 (GraphPad) using scramble siRNA transfected cells as control. Moreover, 70 µl cells (10⁶ cells/ml) were cultured with DMEM medium containing 1% FBS into 0.5% Matrigel coated transwell (657,638, Greiner Bio-One, UK). The invaded cells on the bottom side of the insert chamber were fixed with 3.7% paraformaldehyde and stained with 2% crystal violet. The stained cell colonies were imaged under a microscopy (490042-0002-000, ZEISS Axioscope, Carl Zeiss, München, Germany) and counted for cell invasion ability with image J software (National Institutes of Health, USA).

Formalin-fixed paraffin-embedded (FFPE) tumour samples were available from four patients (cutaneous SCC, nodal melanoma metastasis, subcutaneous melanoma metastasis, Merkel cell carcinoma), before ICI initiation. Serial sections were cut and stained with haematoxylin and eosin for routine diagnostics. After deparaffinization and head-induced antigen retrieval with citrate pH6 retrieval buffer, immunohistochemical (IHC) stainings were performed. Sections were incubated at room temperature for 1 h with an anti-collagen XVII antibody (Abcam, Cambridge, UK). Sections were analysed using the Dako REAL™ detection system, alkaline phosphatase/RED, Rabbit/Mouse (Dako, Glostrup, Denmark). Pictures of stainings were taken with a microscope Zeiss Axioscope. For visualization, the program AxioVision (Zeiss, Jena, Germany, SE64 release 4.9) was used.