Axio imager m2m

Fluorescence images of brain slices were taken using a Zeiss fluorescent microscope (Axio Imager M2M). Confocal micrographs were acquired with either an Olympus Fluoview FV1000 or a Zeiss LSM880 AiryScan confocal microscope. For analysis, every 10th brain section approx. 1 mm anterior and posterior to the injection site were immunostained, with an interval of 250 μm per section. The definition of the CC was based on the mouse brain atlas (Franklin and Paxinos, 2019 ).
Total Aβ load was determined by calculating the relative areal fraction occupied with Aβ-positive staining (in %) in the CC using the image analysis software ImageJ (National Institutes of Health freeware, immunoblot analysis of injected brain homogenates). About four to seven animals per group and four to five sections per animal were analyzed.
Cell number was quantified by counting the number of positively labeled cells in the CC of the animals. This was mostly done in a semi-automated manner using custom-written macros. Since GFAP exclusively stains the processes of reactive astrocytes, the assessed cell number was substituted by the relative areal fraction occupied with the GFAP-positive signal (in %). To measure the thickness of the CC, six to seven animals per group and three to five sections per animal were analyzed. All analyses were conducted in a blinded manner.

Microscopy images were taken to identify filler distributions, changes in particle size and potential processing defects. Reflected light microscopy images were taken using an AxioImagerM2m from Zeiss AG (Oberkochen, Germany). Samples were prepared by embedding them in a transparent epoxy resin, with subsequent grounding and polishing.

An image of the entire cell array area was obtained using a fluorescence microscope (Axio Imager M2m; Carl Zeiss, Oberkochen, Germany) integrated with a 10× objective lens and a computer-operated motorized stage, a digital camera (AxioCam 503 mono; Carl Zeiss), and ZEN image acquisition software (Carl Zeiss). We defined a CTC as a DAPI-positive, cytokeratin-positive, and CD45-negative cell. The number of CTCs in 3 mL of peripheral blood was normalized to that in 7.5 mL to compare with the CellSearch system.

For microstructural studies, specimens were polished and etched in a 5 vol% nital solution. Microstructure was examined by an optical microscope (Axio Imager M2M, Zeiss, US).

Scanning electron microscopy (SEM) images
were acquired using a Zeiss Ultra Plus 55. Imaging was performed at
an accelerating voltage of 5 kV, with an InLens secondary electron
(SE) detector and a working distance of 4 mm. Energy dispersive X-ray
spectroscopy (EDS) elemental maps were acquired using a 50 mm² silicon drift EDS detector from Oxford instruments. The freely
available software ImageJ was used for size analysis and quantification
of the metal catalyst coverage. A Leica S8 APO stereo microscope was
used at a magnification of 80× for obtaining the Si substrate
light microscopy photographs. Zeiss Axio Imager M2m using a 100×
objective was used for imaging the patterned silicon samples.

Following rehydration, 4 µm thick sections were stained with rabbit polyclonal chromogranin A (chgA; a marker of total enteroendocrine cells) antibody (diluted 1/500; 20085; Immunostar, Hudson, WI, USA). After incubation with a secondary biotinylated goat anti-rabbit antibody (diluted 1/1000, A24541; Life Technologies, Carlsbad, CA, USA), chgA was detected with Alexa Fluor 568-conjugated Streptavidin (S11226; Invitrogen, Waltham, MA, USA). Sections were mounted using ProLong Gold Antifade Reagent (Invitrogen) containing DAPI to counterstain the nuclei. The density of CCK-producing cells that were stained green (endogenous GFP) and positive for chgA was measured by fluorescence microscopy (Axio Imager M2m; Zeiss, Iéna, Germany) in the crypts of three duodenal sections under a 40× objective. The data were expressed as the percentage of chgA-positive cells or CCK (GFP)-positive cells per crypt.

Fluorescence images of brain slices were taken using a Zeiss fluorescent microscope (Axio Imager M2M). For analysis every tenth brain section of a single hemisphere was immunostained. Areas such as the olfactory bulb, mitral cell layer, piriform cortex and the SVZ were defined based on the mouse brain atlas [38 ]. Total Aβ load was determined by calculating the % areal fraction occupied by Aβ positive staining in the olfactory bulb using the imaging software ImageJ (National Institutes of Health freeware). 5–6 animals per group and 6 sections per animal were analyzed. The sections represented always the same layers in each animal, starting from Bregma 5.0 to Bregma 3.7.
Cell number was quantified by counting the number of positive labeled cells in the area of interest of the animals. 5–7 animals per group and 3–4 sections per animal were analyzed. The 25 µm thick serial coronal sections represented always the same layers in each animal, starting from Bregma 5.0 to Bregma 3.7. The sections for the piriform cortex were chosen between Bregma 2.3 and 1.6.
Cell counting was done in the olfactory bulb and the area of the olfactory bulb was measured with the ImageJ software. Cell counts were performed within a defined volume based on the region of interest and the thickness of the section (25 µm). All analyses were conducted in a blinded manner.