Axiocam mrc digital camera

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.

A Zeiss M2 Imager microscope (Carl Zeiss, Jena, Germany) connected to an AxioCam Mrc digital camera was used for brightfield microscopy (α-Syn, phospho-α-Syn and TH). Images were collected through a 63X lens for the α-Syn and phospho-α-Syn images, and a 20X lens for the TH images, using Microlucida software (v2019.1.3; MBF Bioscience, VT, USA). Fluorescence analysis was performed for α-Syn and Iba1 using a Leica SP5-II confocal microscope (Leica Microsystems, Wetzlar, Germany). 40X and 60X lenses were used, and Z sectioning was performed at 1-2 μm intervals in order to verify the co-localization of markers. Image extraction and analysis was conducted via the Leica LAS software (LAS AF 2.7.3.9723).

After METH administration and Ag sensitization, each mouse was euthanized and lung/spleen tissues were excised and fixed in 4% paraformaldehyde (Sigma) for 24 h. Tissues were processed, embedded in paraffin, and 4 μm sagittal sections were fixed to glass slides. The tissues were then subjected to hematoxylin and eosin (H&E) staining, CD4- (clone W3/25)^{44 (link)}, and CD8- (clone 32-M4)^{45 (link)} specific antibody (Ab; conjugated to horseradish peroxidase; dilution: 1:1000; Santa Cruz Biotechnology; SCB) immunostaining to assess tissue morphology, helper T cell, and cytotoxic T cell infiltration (brown), respectively. The slides were visualized using an Axiovert 40 CFL inverted microscope (Carl Zeiss; CZ), and images were captured with an AxioCam MRc digital camera using the Zen 2011 digital imaging software. The hyperplasia of type II pneumocytes was recorded microscopically in single cuboidal cells showing large nuclei, prominent nucleoli, and scant to abundant cytoplasm. The infiltration of CD4⁺ and CD8⁺ cells to pulmonary and splenic tissue was quantified by cell counts using the recorded 40 × images. Each image was blindly analyzed by three independent investigators. Ten microscopic fields per image were counted and the average was calculated per image (n = 10 images; 10 fields per image; 2 images per mouse; 5 mice per condition).

Light microscopic examination was performed on plastic embedded thick sections from 4% PFA fixed posterior sections from eyes of the affected male (LAB4) and his heterozygous sibling (LAB6), as well as from an unaffected 10-year-old German spaniel dog. The samples were post-fixed in 2.5% glutaraldehyde-2% formaldehyde (2 hours), 2% glutaraldehyde-1% osmium tetroxide (1.5 hours), and 2% osmium tetroxide (1.5 hours). The posterior segments were then trimmed into segments 2–5 mm in length, taken from the superior retina (three sections located 0.5 cm to 1.5 cm dorsal to the optic nerve), and the nasal retina (two sections from non-tapetal retina). These were dehydrated, and embedded in epoxy resin (PolyBed 812; Polysciences, Warrington, PA). Tissues were sectioned at 1μm and stained with azure II-methylene blue/paraphenylenediamine counterstain. Sections were examined with a 40× objective on a light microscope (Axioplan; Carl Zeiss Meditec GmbH Oberkochen, Germany) and images collected with an AxioCam MrC digital camera (Carl Zeiss Meditec GmbH Oberkochen, Germany).

Thin smears of parasites were fixed with 4% paraformaldehyde for 30 min at room temperature. The fixed cells were permeabilized with PBS containing 0.1% Triton X-100 for 5 min followed by blocking in 3% BSA (w/v) in PBS at 4 °C overnight. Primary antibodies were diluted as required; species-specific Alexa Fluor 488- and 594-conjugated secondary antibodies (Life Technologies, Inc.) were used to visualize primary antibody binding. All antibody dilutions were carried out with 3% BSA (w/v) in PBS. Slides were mounted for microscopic examination using Prolong Gold antifade reagent with DAPI (Life Technologies, Inc.). Slides were viewed using a Zeiss Axioplan 2 microscope, and images were captured using a Zeiss Axiocam MRc digital camera and AxioVision 4.8.2 software and prepared for publication using Adobe Photoshop.
To image stages of invasion of erythrocytes, tightly synchronized PfMyoB-GFP-expressing late stage schizonts were mixed with erythrocytes, and samples were taken after 2, 5, 8, 10, and 30 min and fixed in solution with 4% paraformaldehyde, 0.01% glutaraldehyde for 60 min at room temperature. Further processing was carried out as described previously (23 (link)).

At days 3 and 7 post-infection, wounded tissues were excised from euthanized mice; the tissues were fixed in 10% formalin and embedded in paraffin. Four micrometer vertical sections were cut and then fixed to glass slides and subjected to Haematoxylin and Eosin (H&E), Gram, MPO, or collagen type I (Santa Cruz Biotechnology, Dallas, TX, USA) mAb staining to assess morphology, bacterial burden, neutrophil infiltration, or collagen deposition, respectively. The slides were visualized using an Axiovert 40CFL inverted microscope (Carl Zeiss, Thornwood, NY, USA), and images were captured with an AxioCam MrC digital camera using the Zen 2011 digital imaging software. Quantification of the collagen staining was carried out using ImageJ software using threshold filters to isolate the stain and measurement of colorimetric intensity.

Immunohistochemical analysis was performed on a thick (5 μm), paraformaldehyde-fixed, paraffin-embedded tumor tissue sections using the Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC Kit (Abcam Inc., Cambridge, United Kingdom) as reported precedingly (Wang et al., 2012 (link)). Briefly, tissue sections were immunostained with an anti-Ki-67 (1:50) or anti-PCNA (dilution 1:200). The number of Ki-67 or PCNA positive cells was determined in 10 randomly selected microscopic fields at ×400 magnification. The proliferation index of Ki-67 or PCNA was calculated as: the number of Ki-67 or PCNA positive cells/the total cell count × 100%. Immunofluore analyses for Ki67 (dilution 1:50) and PCNA (dilution 1:200) were carried out with the Vecta Elite kit (Vector Laboratories, Burlingame, CA, United States) based on the manufacturer’s manual. Sample analysis and image acquisition were achieved by Axio Scope A1 fluorescence microscope which was equipped with AxioCam MRC digital camera (Carl Zeiss, Oberkochen, Germany).