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1

Histochemical and TUNEL Analysis of ca10a and ca10b Morphants

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To study the tissue morphology of 5 dpf ca10a and ca10b morphant larvae a histochemical analysis was performed. Prior to the analysis the larvae were washed with PBS and fixed in 4% paraformaldehyde (PFA) in PBS for 3 hours at room temperature and the fixed larvae were transferred to 70% ethanol and stored at 4°C before being embedded in paraffin. The paraffin embedded samples were sectioned into 5 μm slices for the histochemical staining.
The fixed sections were deparaffinized in xylene, rehydrated in an alcohol series and histologically stained with Mayer's Hematoxylin and Eosin Y (both from Sigma-Aldrich). After dehydration, the slides were mounted with Entellan Neu (Merck; Darmstadt, Germany), examined and photographed using a Nikon Microphot microscope (Nikon Microphot-FXA, Japan).
To detect apoptotic cell death in the ca10a and ca10b morphants and the control larvae, a TdT-UTP nick end labeling (TUNEL) assay was performed for the prepared slides using the QIA39 FragEL DNA Fragmentation Detection Kit (Merck Chemicals Ltd., Nottingham United Kingdom). Briefly, the deparaffinized sections of the larvae were incubated with the TdT enzyme followed by incubation with anti-digoxigenin. Fluorescence staining was detected and photographed using a Nikon Microphot microscope (Nikon Microphot-FXA, Japan).

Aspatwar A., Tolvanen M.E., Ojanen M.J., Barker H.R., Saralahti A.K., Bäuerlein C.A., Ortutay C., Pan P., Kuuslahti M., Parikka M., Rämet M, & Parkkila S. (2015). Inactivation of ca10a and ca10b Genes Leads to Abnormal Embryonic Development and Alters Movement Pattern in Zebrafish. PLoS ONE, 10(7), e0134263.

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2

Immunocytochemistry of Cultured Cells

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PMEC were seeded on 12 mm coverslips (Carl Roth) in 24-well plate (Biochrom) at a density of 10 000 cells/well. After two days of culturing, medium was discarded, and coverslips were washed twice with PBS and fixed with ice-cold methanol (−20 °C, Carl Roth) for 20 min. Cells were permeabilized with 0.2% Triton X-100 (Carl Roth), diluted with PBS for 5 min and washed twice with PBS. Non-specific binding sites were blocked by incubating the coverslips with 10% FBS in PBS for 30 min at room temperature. Coverslips were washed twice with PBS and incubated with mouse anti-cytokeratin 18-fluorescein isothiocyanate (anti-Cy18-FITC, Sigma-Aldrich) and mouse anti-alpha-smooth muscle actin antibodies (clone 1A4, Sigma-Aldrich), respectively in a humidified chamber for 1 h. Coverslips were washed three times with PBS. Bound anti-alpha-smooth muscle actin antibody was visualized by 1 h incubation of the coverslips with goat anti-mouse FITC-labeled secondary antibody (Sigma-Aldrich). Nuclei of the cells were stained with 4’,6-diamidino-2-phenylindole (DAPI, Carl Roth) for 15 min. Coverslips were washed twice with PBS, air dried and mounted with 1,4-diazabicyclo[2.2.2]octane (DABCO) on glass slides (both from Carl Roth). Coverslips were analyzed by immunofluorescence microscopy (Microphot-FXA, Nikon, Düsseldorf, Germany).

Jaeger A., Bardehle D., Oster M., Günther J., Muráni E., Ponsuksili S., Wimmers K, & Kemper N. (2015). Gene expression profiling of porcine mammary epithelial cells after challenge with Escherichia coli and Staphylococcus aureus in vitro. Veterinary Research, 46(1), 50.

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3

Intrahippocampal Gingipains Induce Neurodegeneration

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Example 2

Adult male mice (CD-1, 25 g approximately) n=6 per group were anaesthetized and injected unilaterally intrahippocampally using standard stereotaxic techniques. Gingipains RgpB and Kgp, purified from P. gingivalis were diluted prior to injection to 10 μg/ml. Seven days post-surgery the animals were anaesthetized, perfused and humanely killed and brains removed and sectioned for histological analysis. Fluoro-Jade staining was then be performed on sections of hippocampus to assess for neurodegeneration (Schmued L C and Hopkins K J, 2000). Fluoro-Jade staining identifies cell bodies, dendrites, axons and axons terminals of degenerating neurons but does not stain healthy neurons, myelin, or vascular elements.

Brain sections were examined with an epifluorescence microscope (Nikon Microphot FXA) using a filter system suitable for visualizing fluorescein or fluorescein isothiocyanate (FITC). Images were acquired with a Leica DC Camera and an Image Analysis software (Leica IM50). Fluoro-Jade C-positive degenerating neurons appeared bright yellow-green against a dark background and were clearly identified in the animal groups treated with Gingipains. No Fluoro-Jade C-positive cells were observed in vehicle-treated group (FIG. 4).

US11332464B2. Inhibitors of lysine gingipain (2022-05-17). Cortexyme, Inc. [US]. Inventors: Andrei W. Konradi [US], Stephen S. Dominy [US], Casey Crawford Lynch [US], Craig Coburn [US], Joseph Vacca [US].

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4

Histological Analysis of SARDS Canine Retina

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Three eyes from SARDS patients and 5 eyes from healthy control dogs were fixed in 10% formalin. Eyes were embedded in paraffin and 7‐μm tissue sections were prepared. A total of 10 retinal sections of central (temporal and nasal) and peripheral (temporal and nasal) retina were evaluated for each eye. Standard hematoxylin and eosin stain was performed and slides were coverslipped. Tissue sections were examined under a photomicroscope (Microphot FXA; Nikon, New York, NY). Images were captured using a camera (Megaplus, model 1.4; Eastman Kodak, Rochester, NY) connected to a frame grabber (MegaGrabber; Perceptics, Knoxville, TN) in a computer (Macintosh 8100/80 AV; Apple Computer, Cupertino, CA) using image acquisition and analysis software (Metamorph; Molecular Devices, Sunnyvale, CA).

Grozdanic S.D., Lazic T., Kecova H., Mohan K, & Kuehn M.H. (2018). Optical coherence tomography and molecular analysis of sudden acquired retinal degeneration syndrome (SARDS) eyes suggests the immune‐mediated nature of retinal damage. Veterinary Ophthalmology, 22(3), 305-327.

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5

Leaf Structure Analysis by SEM and Microscopy

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For scanning electron microscopy (SEM), small pieces of fresh- and air-dried leaves were fixed to sample holders and observed with a scanning electron microscope (TM 3030 Plus Hitachi).
The cuticular membrane was observed in a thin leaf cross section by optical microscopy. The leaves were impregnated with DP 1500 polyethylene glycol and cross sections of approximately 10 µm thickness were cut with a rotary microtome (Medite M530). The sections were stained with safranin and astral blue and were mounted in Euparal. Observations were made using a light microscope (Leica DM LA) and the photomicrographs were taken with a Nikon Microphot-FXA.

Simões R., Rodrigues A., Ferreira-Dias S., Miranda I, & Pereira H. (2020). Chemical Composition of Cuticular Waxes and Pigments and Morphology of Leaves of Quercus suber Trees of Different Provenance. Plants, 9(9), 1165.

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6

Assessing Neurodegeneration in Gingipain-Treated Mice

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Example 2

Adult male mice (CD-1, 25 g approximately) n=6 per group were anaesthetized and injected unilaterally intrahippocampally using standard stereotaxic techniques. Gingipains RgpB and Kgp, purified from P. gingivalis were diluted prior to injection to 10 μg/ml. Seven days post-surgery the animals were anaesthetized, perfused and humanely killed and brains removed and sectioned for histological analysis. Fluoro-Jade staining was then be performed on sections of hippocampus to assess for neurodegeneration (Schmued L C and Hopkins K J, 2000). Fluoro-Jade staining identifies cell bodies, dendrites, axons and axons terminals of degenerating neurons but does not stain healthy neurons, myelin, or vascular elements.

Brain sections were examined with an epifluorescence microscope (Nikon Microphot FXA) using a filter system suitable for visualizing fluorescein or fluorescein isothiocyanate (FITC). Images were acquired with a Leica DC Camera and an Image Analysis software (Leica IM50). Fluoro-Jade C-positive degenerating neurons appeared bright yellow-green against a dark background and were clearly identified in the animal groups treated with Gingipains. No Fluoro-Jade C-positive cells were observed in vehicle-treated group (FIG. 4).

US09988375B2. Inhibitors of lysine gingipain (2018-06-05). Cortexyme, Inc. [US]. Inventors: Andrei Konradi [US], Stephen S. Dominy [US], Casey Crawford Lynch [US], Craig Coburn [US], Joseph Vacca [US].

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7

Macrophage Migration Assay with RBCs

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bEnd.3 cells (murine endothelial cells, ATCC) were grown to confluence on 3.0-μm pore size 24-well inserts (BD Biosciences); 1×10⁷ packed RBCs from chow diet (CD)–fed (CD-RBC) or HFD-fed (HFD-RBC) mice in defined medium were added to the bottom of the wells. Then, J744.1 cells (murine monocyte/macrophage cell line, ATCC) were added to the inserts and allowed to adhere for 1 hour at 37°C, 5% CO₂. Nonadherent cells were removed by aspiration, and the inserts were filled with fresh defined medium. Migration proceeded for 16 hours at 37°C, 5% CO₂. Inserts were excised and fixed in ice-cold methanol, after which nonmigrated macrophages on the luminal side of the inserts were removed by using a cotton swab, whereas transmigrated macrophages on the abluminal side were preserved by placing the inserts on slides and mounted by using Vectashield/DAPI (Vector Labs). Images were captured using fluorescence (Nikon Microphot-FXA, 10×, n=5 representative fields per insert), and results were analyzed using Image J (National Institutes of Health).

Unruh D., Srinivasan R., Benson T., Haigh S., Coyle D., Batra N., Keil R., Sturm R., Blanco V., Palascak M., Franco R.S., Tong W., Chatterjee T., Hui D.Y., Davidson W.S., Aronow B.J., Kalfa T., Manka D., Peairs A., Blomkalns A., Fulton D.J., Brittain J.E., Weintraub N.L, & Bogdanov V.Y. (2015). Red Blood Cell Dysfunction Induced by High-Fat Diet: Potential Implications for Obesity-Related Atherosclerosis. Circulation, 132(20), 1898-1908.

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8

Transverse Microscopy of Plant Tissues

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A sample of each tissue was impregnated with DP1500 polyethylene glycol, and transverse microscopic sections of approximately 17 μm thickness were cut with a microtome (Leica SM 2400). The cork and phloem sections were stained with triple staining astra blue/crysoidine/sudan IV, and the xylem sections with safranin. All the sections were observed in a light microscopic using Leica DM LA and photomicrographs were taken with a Nikon Microphot-FXA.

Lourenço A., Rencoret J., Chemetova C., Gominho J., Gutiérrez A., del Río J.C, & Pereira H. (2016). Lignin Composition and Structure Differs between Xylem, Phloem and Phellem in Quercus suber L.. Frontiers in Plant Science, 7, 1612.

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9

In Vivo Spleen Imaging of RBCs

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One hundred μL of packed CD-RBC or HFD-RBC were labeled with 15 μmol/L Cell-Tracker Red CMTPX fluorescent dye (Invitrogen) for 30 minutes at 37°C, washed with PBS, and suspended in defined medium at 50% hematocrit. Two hundred μL of RBCs were infused via retro-orbital route into CD-fed mice, and after 24 hours the animals were anesthetized and euthanized by cardiac puncture/perfusion with PBS. Spleens were then isolated, rinsed in PBS, embedded in optimum cutting temperature medium crosswise that included 4 to 5 cut segments per spleen, snap-frozen, and stored at −80°C. With the use of a cryotome, 10-μm sections were cut (≈50 sections per each spleen), fixed with 10% paraformaldehyde, and visualized under fluorescence (Nikon Microphot-FXA). Sections were first scanned at low power (1.5× magnification), and then images were captured for analysis at 100× magnification. At least 3 representative fields for each section of each spleen were examined such that images were obtained to cover the entire cross section of the spleen. Images were uniformly contrast adjusted to reduce background (autofluorescence), converted to binary, and analyzed with Image J software to determine cell number (fluorescent signal density over surface area). Events with pixel sizes >100 were deemed true cell events.

Unruh D., Srinivasan R., Benson T., Haigh S., Coyle D., Batra N., Keil R., Sturm R., Blanco V., Palascak M., Franco R.S., Tong W., Chatterjee T., Hui D.Y., Davidson W.S., Aronow B.J., Kalfa T., Manka D., Peairs A., Blomkalns A., Fulton D.J., Brittain J.E., Weintraub N.L, & Bogdanov V.Y. (2015). Red Blood Cell Dysfunction Induced by High-Fat Diet: Potential Implications for Obesity-Related Atherosclerosis. Circulation, 132(20), 1898-1908.

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10

Gingipain-Induced Hippocampal Neurodegeneration

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Example 2

Adult male mice (CD-1, 25 g approximately) n=6 per group were anaesthetized and injected unilaterally intrahippocampally using standard stereotaxic techniques. Gingipains RgpB and Kgp, purified from P. gingivalis were diluted prior to injection to 10 μg/ml. Seven days post-surgery the animals were anaesthetized, perfused and humanely killed and brains removed and sectioned for histological analysis. Fluoro-Jade staining was then be performed on sections of hippocampus to assess for neurodegeneration (Schmued L C and Hopkins K J, 2000). Fluoro-Jade staining identifies cell bodies, dendrites, axons and axons terminals of degenerating neurons but does not stain healthy neurons, myelin, or vascular elements.

Brain sections were examined with an epifluorescence microscope (Nikon Microphot FXA) using a filter system suitable for visualizing fluorescein or fluorescein isothiocyanate (FITC). Images were acquired with a Leica DC Camera and an Image Analysis software (Leica IM50). Fluoro-Jade C—positive degenerating neurons appeared bright yellow-green against a dark background and were clearly identified in the animal groups treated with Gingipains. No Fluoro-Jade C—positive cells were observed in vehicle-treated group (FIG. 4).

US10301301B2. Inhibitors of lysine gingipain (2019-05-28). Cortexyme, Inc. [US]. Inventors: Andrei Konradi [US], Stephen S. Dominy [US], Casey Crawford Lynch [US], Craig Coburn [US], Joseph Vacca [US].

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Microphot fxa

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55 protocols using microphot fxa

Histochemical and TUNEL Analysis of ca10a and ca10b Morphants

Immunocytochemistry of Cultured Cells

Intrahippocampal Gingipains Induce Neurodegeneration

Histological Analysis of SARDS Canine Retina

Leaf Structure Analysis by SEM and Microscopy

Assessing Neurodegeneration in Gingipain-Treated Mice

Macrophage Migration Assay with RBCs

Transverse Microscopy of Plant Tissues

In Vivo Spleen Imaging of RBCs

Gingipain-Induced Hippocampal Neurodegeneration

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