Axioscop 2 plus

All retinal wholemounts were examined and photographed with a fluorescence microscope (Axioscop 2 Plus; Zeiss, Jena, Germany) equipped with a digital camera (ProgRes C10; Jenoptic, Jena, Germany) and a computer driven motorized stage (ProScan H128 Series; Prior Scientific Instruments, Cambridge, UK) controlled by Image-Pro Plus software (IPP 5.1 for windows; Media Cybernetics, Silver Spring, MD, USA), following protocols that are standard in our laboratory [15 (link),25 (link),35 (link)]. Photomontages of wholemounts were constructed from 90 consecutive frames captured side by side and, if necessary, images were further processed with a graphics editing programme (Adobe Photoshop CS 8.0.1; Adobe Systems, Inc., San Jose, CA, USA).

Paraffin blocks were sectioned to 4 µm, deparaffinized and dehydrated. Afterwards the sections were stained with hematoxylin and eosin using convectional protocol. The slides were visualized with Axioscop 2 PLUS (Carl Zeiss, GmbH, Jena, Germany) at the Multiple-access Center for Microscopy of Biological Subjects (Institute of Cytology and Genetics, Novosibirsk, Russia).

The slides were deparaffinized in xylene, rehydrated in grade ethanol and washed with 0.1% tween in PBS (PBS-T). For antigen retrieving, the slides were heated to 121 °C in an autoclave for 20 min in a citrate buffer (10 mmol/L, pH 6.0). The endogenous peroxidase was blocked by immersing the slides in 3% H₂O₂ for 10 min. Subsequently, the slides were incubated with anti-C-MYC primary antibodies (MAB36961, R&D systems) at 4 °C overnight. After washing, each slide was incubated with HRP-conjugated anti-rabbit secondary antibodies (#31460, Thermo Fischer Waltham, MA, USA). Afterwards, the slices were incubated with the 3,39-diaminobenzidine tetrahydrochloride (DAB) reagent for 10 min. Then, hematoxylin re-staining was performed, followed by dehydration, clearing and mounting. For the negative control, slides were incubated with PBS -T instead of primary antibodies. The slides were visualized with Axioscop 2 PLUS (Carl Zeiss, GmbH, Jena, Germany) at the Multiple-access Center for Microscopy of Biological Subjects (Institute of Cytology and Genetics, Novosibirsk, Russia).

Morphometric analyses were carried out with an Axioscop 2plus epifluorescence microscope (Zeiss, Oberkochen, Germany) equipped with a cooled color charge-coupled device camera (CCD; Axiovision Cam, Zeiss) interfaced with an interactive and automatic image analyser (Axiovision, Zeiss). Serial tissue sections were analyzed under 200 × magnification. The quantification of digitised signals was completed using a semi-automated algorithm of the image analysis software KS300 (Zeiss), as previously described (Martelli et al. 2009 (link); Barboni et al. 2012b (link)). Guided programs (macros for KS300) were created to count internally a standard field of 15 000 μm²:
In particular, the quantification of the digitised fluorescent signals was accomplished after the background densitometry calibration, the algorithm detected and separated the fluorescent regions of interest from their background and created a new image on the basis of their gray values. Thus, the process created a binary image from true color images. The analyses were performed on the whole area of the cross-section (Martelli et al. 2009 (link); Barboni et al. 2012b (link)).
The analyses were carried out on at least three serial sections/calcaneal tendons and at least two different fields/sections for each analysis.