ApoE KO mice fed with a high-fat diet for 4 months and age-matched wild-type mice were intravenously administered with 0.5 mg of the various DiD-loaded nanoparticles via the tail vein. The nanoparticle dosage was chosen based on previous studies using PNPs to target vascular injury.29 (link) After allowing the nanoparticles to distribute for 24 hours, the mice were euthanized and then perfused with PBS to remove unbound nanoparticles. The aortas were isolated for imaging using a Keyence BZ-9000 fluorescence microscope. Oil Red O staining was performed to confirm the formation of atherosclerotic plaque in the ApoE KO mice. Quantification of fluorescence intensity was carried out using ImageJ software. For histological analysis, aortas from PNP-administered ApoE KO mice were collected and embedded in OCT compound for cryosectioning. Sections were then fixed in 10% formalin for 30 minutes and blocked with 4% bovine serum albumin (Sigma Aldrich). For fluorescent imaging, sections were incubated with anti-mouse CD54 (YN1/1.7.4; Biolegend), anti-mouse CD68 (FA-11; Biolegend) or polyclonal anti-collagen IV (ab6586; Abcam) followed by an Alexa Fluor 555-conjugated secondary (Biolegend). After washing three times with PBS containing 0.05% Tween 20, the sections were mounted with DAPI-containing mounting medium. All images were taken using a Keyence BZ-9000 fluorescence microscope.
Bz 9000 fluorescence microscope
Manufactured by Keyence
Sourced in Japan, Germany, United States
The Keyence BZ-9000 is a fluorescence microscope. It is designed to capture high-quality images by utilizing fluorescence technology.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
Bz 2 analyzer, by Keyence (6 mentions)
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Bz 2 analyzer software, by Keyence (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (5 mentions)
Vectashield, by Vector Laboratories (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (3 mentions)
Target retrieval solution, by Agilent Technologies (3 mentions)
Hybrid cell count software, by Keyence (3 mentions)
Vectashield mounting medium, by Vector Laboratories (3 mentions)
Paraformaldehyde (pfa), by Fujifilm (3 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Normal goat serum (ngs), by Agilent Technologies (2 mentions)
236 protocols using bz 9000 fluorescence microscope
1
Nanoparticle-Mediated Atherosclerosis Imaging
2
Immunofluorescence Staining of ARPE-19 and sNF96.2 Cells
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ARPE-19 cells cultured in 96-well plates were fixed for 30 min at room temperature with 4% paraformaldehyde in PBS, washed with PBS, and exposed for 1 h at room temperature to 3% BSA in PBS before incubation overnight at 4 °C with biotinylated HABP (5 µg/ml, Seikagaku) and 3% BSA in PBS. The cells were then washed with PBS, incubated for 60 min at room temperature with streptavidin–Alexa Fluor 488 (Invitrogen) and Hoechst 33342 in PBS containing 3% BSA, washed again with PBS, and imaged with a BZ-9000 fluorescence microscope (Keyence).
sNF96.2 cells cultured in 35-mm dishes were fixed for 15 min at room temperature with 4% paraformaldehyde in PBS, washed with PBS, and then permeabilized for 30 min at room temperature with 0.2% Triton X-100 in PBS. The cells were stained for 90 min at room temperature with antibodies to collagen type III (Abcam) diluted in PBS containing 1% BSA, washed with 0.2% Triton X-100 in PBS, incubated for 60 min at room temperature with Alexa Fluor 594–conjugated secondary antibodies (Invitrogen) in PBS containing 1% BSA, and washed again with 0.2% Triton X-100 in PBS. They were finally stained for 5 min with Hoechst 33342 in PBS containing 1% BSA, washed with 0.2% Triton X-100 in PBS, and imaged with a BZ-9000 fluorescence microscope (Keyence).
sNF96.2 cells cultured in 35-mm dishes were fixed for 15 min at room temperature with 4% paraformaldehyde in PBS, washed with PBS, and then permeabilized for 30 min at room temperature with 0.2% Triton X-100 in PBS. The cells were stained for 90 min at room temperature with antibodies to collagen type III (Abcam) diluted in PBS containing 1% BSA, washed with 0.2% Triton X-100 in PBS, incubated for 60 min at room temperature with Alexa Fluor 594–conjugated secondary antibodies (Invitrogen) in PBS containing 1% BSA, and washed again with 0.2% Triton X-100 in PBS. They were finally stained for 5 min with Hoechst 33342 in PBS containing 1% BSA, washed with 0.2% Triton X-100 in PBS, and imaged with a BZ-9000 fluorescence microscope (Keyence).
3
Histology and Immunofluorescence of EPC2 ALI Cultures
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Formalin‐fixed, paraffin‐embedded EPC2 ALI cultures were cut into 5 µm sections and deparaffinized using xylene followed by graded ethanol washes. For histology, sections were stained in hematoxylin, rinsed in tap water and then stained in eosin, followed by dehydration in graded ethanol washes and xylene before mounting with Pertex (Histolab, Askim, Sweden) and xylene (1:1). For immunofluorescence, heat‐induced antigen retrieval in sodium citrate buffer (10 mM citric acid, pH 6.0) was used on deparaffinized sections, and endogenous peroxidase activity was quenched using 3% H2O2 in methanol. After rinsing in 0.2% Tween in PBS, sections were blocked in 3% BSA in PBS containing 5% normal goat serum (Dako, Jena, Germany) for 90 minutes, followed by overnight incubation at 4℃ with rabbit anti‐DSG1 (1 µg/ml; Abcam, Cambridge, UK, cat. no. ab209490) or rabbit anti‐FLG (1 µg/ml; Abcam, cat. no. ab234406). Sections were rinsed and incubated for 1 hour with goat anti‐rabbit AF594 (10 µg/ml; Invitrogen, Carlsbad, CA, USA, cat. no. A11072). Sections were mounted with ProLong™ Gold antifade reagent with DAPI (Invitrogen) for nuclei staining. Immunofluorescent images were acquired using the Keyence Fluorescence Microscope BZ‐9000, and immunofluorescence intensity was quantified using ImageJ software.
4
Immunofluorescence and DNA Damage Assay
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Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100/phosphate-buffered saline (PBS), and then incubated with primary antibodies and coupled with Alexa Fluor secondary antibody. Images were acquired by a Leica True Confocal Scanning (TCS) SP8 microscope system (Leica Microsystems, Wetzlar, Germany) or Fluorescence Microscope BZ-9000 (Keyence, Osaka, Japan). Colocalization analysis with endosome markers was performed with Coloc 2, Fiji’s plugin of ImageJ.29 (link) For DNA damage assessment, cells incubated with AuNPs were fixed and stained with anti-γ-H2AX 12 h after IR. The number of γ-H2AX foci per nucleus was counted in ImageJ. At least 50 nuclei were analyzed in each independent experiment.
5
Immunofluorescence Analysis of Cellular Structures
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Cells were fixed with paraformaldehyde (3.5% in PBS) for 10 min and then permeabilized with 0.5% Triton X-100 in PBS for 10 min. After washing three times with PBS, cells were blocked with 1% BSA in PBS for 1 h at room temperature and washed once. Tissue sections were fixed with paraformaldehyde. After deparaffinization, sections were incubated with citrate buffer (10 mM) for 10 min in a microwave oven. Primary antibodies include mouse anti-keratin 18 (clone Ks18.04) (Progen), mouse anti-keratin 7 (clone OV-TL 12/30) (DAKO), mouse anti-E-cadherin (ab1416) (Abcam), rabbit anti-N-cadherin (SC-7939) (Santa Cruz), mouse anti-tubulin (clone D66) (Sigma-Aldrich); mouse anti-paxillin (clone 349/Pax) and mouse anti-FAK (focal adhesion kinase) (clone 77/FAK) (BD Biosciences). Secondary antibodies (PromoFluor Fluor® anti-mouse or anti-rabbit) were purchased from Promokine. F-actin was stained with PromoFluor 488 or 555 phalloidin (Promokine). After mounting, slides were viewed using a Nikon Eclipse 80i fluorescent microscope and digital images were taken with Visitron Systems 7.4 Slider camera (Diagnostic Instruments) using Spot Advanced software (Diagnostic Instruments) or Keyence fluorescence microscope BZ9000. The full focus function was used to visualize keratin fibers in different planes of the cell.
6
Cell Seeding Optimization on DMA
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Each field containing 14 × 14 spots (DMA with 1 mm spots), 27 × 27 spots (DMA with 500 µm spots) and 39 × 39 spots (DMA with 350 µm spots) was imaged immediately after seeding using KEYENCE Fluorescence Microscope BZ-9000 (KEYENCE, Osaka, Japan) at 2× magnification using “merge” function of the microscope software BZ II-Viewer (KEYENCE, Osaka, Japan). The initial cell number in the droplets was estimated by manual counting using ImageJ (Version 1.51f, Bethesda, MD, USA) software. Spots were grouped depending on the initial quantity of cells in the droplet. The experiment was repeated 3 times independently, with 9 arrays analysed.
7
Histological Evaluation of Intervertebral Discs
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After fixation and decalcification, IVDs were cut through the center along their median plane. Macroscopic images were taken by an 8‐megapixel iSight camera with 1.5 μm pixel size (Apple, Cupertino, California, USA). IVDs were agitated, paraffinized, and sliced to 8 μm sections. Each explant was stained by hematoxylin/eosin (HE) and 1 g/L Safranin‐O (Merck, Kenilworth, New Jersey, USA) 800 mg/L Fast Green FCF staining (Merck) solution. Multiple images were captured by using a KEYENCE fluorescence microscope BZ‐9000 (KEYENCE, Osaka, Japan) and digitally merged via imaging stitching. Subsequently, all sections were independently scored by 3 researchers (H.S., J.S., and T.N.) according to an adapted version of the canine‐specific Bergknut scoring system, where Safranin‐O/Fast Green staining was used instead of alcian blue/Picrosirius Red staining to assess proteoglycan matrix abundance.29 Finally, tissue explants (Table S1) were similarly stained by HE to screen for histological abnormalities.
8
Histological Analysis of Articular Cartilage
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Paraffin‐ and cryo‐sections were stained by hematoxylin/eosin (HE) or 1 g/L Safranin‐O (Merck, Kenilworth, New Jersey, USA) 800 mg/L Fast Green FCF staining (Merck) solution. Paraffin sections were thereafter captured by KEYENCE fluorescence microscope BZ‐9000 (Keyence Ltd., Japan) and digitally merged via imaging stitching. Cryosections were captured through an Olympus IX70 microscope (Olympus, Tokyo, Japan). Subsequently, for part 1 all sections were independently and blindly scored by two researchers (J.S. & S.T.) according to the rat‐specific classification set out in Lai et al.25 For cryosections in part 2, scores were focused on NP‐size, area, cell number, cell morphology, and NP‐AF border categories.
9
Immunostaining of MSCs in Osteogel
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To evaluate the phenotypes of the MSCs encapsulated within the Osteogel, an immunostaining of CD90 was performed. Cells were cultured in the Osteogel for 7 days, washed with DPBS, then fixed with 4% PFA and blocked with 10% Normal Goat Serum for 2 h. Primary anti-human CD90 antibody (Clone 5E10; Sigma-Aldrich GmbH, Schnelldorf, Germany; 1:100 dilution) was added to the cells overnight at 4 °C. Washed using PBS containing 0.5% Tween 20, Osteogels were incubated with secondary AlexaFluor® 594-conjugated goat anti-mouse IgG antibody (ThermoFisher Inc., Waltham, MA, USA; 1.0 mg/mL, 1:200 dilution) and 4′,6-diamidino-2-phenylindole DAPI (ThermoFisher Inc., Waltham, MA, USA; 1:400 dilution). After intensive washing, the immunofluorescent staining was documented by Keyence BZ-9000 Fluorescence Microscope (Keyence GmbH, Neu-Isenburg, Germany).
10
Immunofluorescence Staining for ucMGP
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Cells on coverglasses were treated with 4% paraformaldehyde (Nacalai Tesque, Inc., Kyoto, Japan) for 10 minutes at room temperature and permeabilized with 0.5% Triton X-100 (Nacalai Tesque, Inc.) for 7 minutes at room temperature. For staining with the anti-ucMGP antibody, cells were fixed in acetone at -20°C for 5 minutes. After washing with PBS three times, cells were treated with blocking buffer (PBS containing 10% FBS) for 30 minutes at room temperature, followed by incubation with the primary antibody diluted in blocking buffer overnight at 4°C or for 1 hour at room temperature. The primary antibodies were diluted 1:50–100, including anti-GGCX (GTX109926; GeneTex, Inc.), anti-58K Golgi (ab27043; Abcam, Cambridge, UK), anti-MGP (sc-271906; Santa Cruz), and anti-ucMGP (#ALX-804-642, mAb (BD4.4); Enzo Life Sciences, Antwerp, Belgium) [35 (link)] antibodies. Samples were reacted with secondary antibodies (1:200) (goat anti-mouse or rabbit IgG (H+L), Alexa Fluor® 488- or 546-conjugated; Thermo Fisher Scientific Inc.) for 30 minutes at room temperature with or without Hoechst (H3570, Life Technologies Japan Ltd, Tokyo, Japan). After washing, samples were mounted in anti-fade solution (fluorescence mounting medium; Dako, Glostrup, Denmark) and images were taken with an OLYMPUS BX50 microscope (OLYMPUS), or a Keyence BZ-9000 Fluorescence Microscope (Keyence, Osaka, Japan).
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