Rm 2155

Intestinal morphology analysis was carried out according to the method described by Choe, et al. [42 (link)]. About 5 cm of the mid-portion of the duodenum, jejunum and ileum was excised carefully and phosphate-buffered saline was used to wash the samples before storing in formalin (10%). Dehydration of the samples was done in an automated tissue processor (Leica ASP 3000, Wetzlar, Germany) for 16 h before using a paraffin-embedding system (Leica RM 2155, Wetzlar, Germany) for sample embedment. A rotary microtome machine (Leica RM 2155, Wetzlar, Germany) was used to cut each sample at 4 μm and the sections were positioned on glass slides and heated at 57 °C to dry. The sections were stained using haematoxylin and eosin and viewed under a light microscope (Leica DM LB2, Wetzlar, Germany) with a fitted digital camera (Leica DFC 295, Wetzlar, Germany). For each six replicates, six villi sections were evaluated per slide per intestinal sample, thus giving a total of 36 measurements per sample. Villi height was measured from the tip of the villi to the villus crypt junction and crypt depth was defined as the depth of the invagination between two villi; both villi height and crypt depth were determined using Image-Pro Plus software as described by Touchette, et al. [57 (link)].

For the evaluation of spermatogenesis, histological analyses were performed on the testes of WT and Dcaf17 KO mice at different postnatal ages (5, 14, 23, 32, 42 and 56 days) as well as on epididymides of sexually mature (8 weeks old) mice (n = 3). The tissues were collected and directly fixed in 4% paraformaldehyde (PFA) or in Bouin’s fixative for overnight at 4 °C. Fixed tissues were washed with distilled water and dehydrated in a series of 70–100% ethanol solutions. Dehydrated tissues were embedded in paraffin and 5 µm thick sections were prepared on glass slides using microtome (Leica RM2155, Leica Biosystems, Buffalo Grove, IL, USA). Paraffin embedded tissue sections were deparaffinized, rehydrated and stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) staining according to standard protocols^{51 (link),52 (link)}. Stained testis and epididymis sections were observed at room temperature using Olympus BX53 (Olympus, Center Valley, PA, USA) optical microscope. Digital images were captured using OLYMPUS DP72 microscope camera (Olympus, Center Valley, PA, USA) and OLYMPUS cellSens Entry 1.6 imaging software (Olympus, Center Valley, PA, USA). Adobe Photoshop software (Adobe Inc., San Jose, CA, USA) and Microsoft PowerPoint software were used to assemble images into figures. No post-acquisition modifications were made to the original images.

The fixed samples of the tissue surrounding the implant with the placeholder included were dehydrated and paraffin-embedded. 5 µm thin slices were cut using a rotary microtome (Leica RM 2155, Leica Biosystems, Germany). Afterwards the slices were stained with hematoxylin–eosin and analyzed systematically using a light microscope (Axioskop 40 with AxioCam MRc digital camera and Zeiss AxioVision software, Carl Zeiss AG, Germany) beginning using the 25fold magnification to get a first overview and to identify the former implantation side. Afterwards, the 200 fold magnification was used to systematically investigate the tissue close to the implant for signs of inflammation or other structural changes. The different samples were compared with one another to detect any differences between the groups or between the sides with the different implants. Identification of nanoparticles themselves was not possible because no additional fluorescence functionalization of the MNPSNP was performed.