Histology slides were scanned at 40× resolution using a TissueScope LE (Huron Digital Pathology, Waterloo, Ontario, Canada). Digital scanning allowed for complete mapping of entire spinal cord sections and clear visualization of margins and anatomical landmarks at an optimal resolution of (0.2 μm/pixel [Px] at 40×) for image analysis. High quality tiff image file (1721 × 985 Px or 3259 × 1174 Px) fields were exported from TissueScope LE and imported in NIH ImageJ 64 VER1.44o (NIH, Bethesda, MD) for analysis. To determine the total area, number, and size of glial cells and neurons, 12 tiff images were analyzed per vervet (6 fields per cervical and 6 fields per lumbar spinal cord segments) (Supplementary Fig. S1 ). Spinal cord tissue sections were blinded and scored manually and compared to automated counting. For automated analysis, an unbiased threshold was applied to regions of interest to determine the morphological findings across treatment groups (Supplementary Fig. S1 ). Slides stained with thioflavine-S and 4′,6-diamidino-2-phenylindole (DAPI) were visualized at 20× magnification using a Zeiss Apotome fluorescent microscope (Thornwood, NY).
Apotome fluorescent microscope
Manufactured by Zeiss
Sourced in Germany, United States
The Apotome fluorescent microscope is a high-performance imaging system designed for advanced fluorescence microscopy. It utilizes a structured illumination technique to provide optical sectioning, allowing for improved image contrast and resolution compared to conventional widefield fluorescence microscopy. The core function of the Apotome is to enable precise and detailed imaging of fluorescently labeled samples.
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Lab products found in correlation
Chicken anti gfp primary antibody, by Abcam (2 mentions)
Alexa fluor 488 conjugated goat anti chicken secondary antibody, by Thermo Fisher Scientific (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
Dp70 camera, by Olympus (2 mentions)
Tx 100, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by HiMedia (1 mentions)
Igf 1, by Thermo Fisher Scientific (1 mentions)
Brdu detection kit, by Roche (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Laminin, by Abcam (1 mentions)
Anti mpo, by Abcam (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Af2089, by R&D Systems (1 mentions)
Sc 6458, by Santa Cruz Biotechnology (1 mentions)
23 protocols using apotome fluorescent microscope
1
Automated Spinal Cord Histology Analysis
2
Immunofluorescence and Histological Analysis of Lung Tissue
3
Lycopersicon esculentum Staining of SDS-BA Scaffold
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Expanded SEJVECs on group II (1% SDS) BA scaffold were fixed in 4% paraformaldehyde (Himedia, Mumbai, India) and stained by FITC-conjugate Lycopersicon esculentum+/+ stain (lectin positive) prepared 2 μg/mL (1 h) (Zeiss, Apotome fluorescent microscope) (Favre et al. 2003 (link)).
4
Myoblast Proliferation Regulation by pPIC Factors
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pPICs and human myoblasts were plated in 24-well plates at a density of 5 × 103 per well and were serum starved for 6 h in 0% serum DMEM/F12 medium. To investigate the effect of pPIC conditioned media on proliferation, wells were either supplemented with standard GM, unconditioned medium (UM), pPIC conditioned medium (CM), or heat-inactivated conditioned medium (ICM). Each well was supplemented with BrdU (1 μg/ml) every 8 h, fixed in 4% paraformaldehyde after 24 h, and BrdU incorporation was then assessed using the BrdU detection kit (Roche) (n = 3/condition).
In order to investigate the effect of growth factors on pPIC proliferation, wells were serum starved for 6 h and then switched to basal media (control), or supplemented with IGF-1 (100 ng/ml; Peprotech), HGF (100 ng/ml; Peprotech), NRG-1 (100 ng/ml; R&D Systems, Minneapolis, Minnesota), or TGF-β1 (5 ng/ml; Peprotech). Each well was supplemented with BrdU (1 μg/ml) every 8 h, fixed after 24 h, and BrdU incorporation was assessed using the BrdU detection kit (Roche) (n = 3/growth factor). Nuclei were counterstained with DAPI. Cells were evaluated using an ApoTome fluorescent microscope (Carl Zeiss). The percentage of BrdUpos cells relative to the total number of cells was determined by counting 5 random fields at ×20 magnification for each dish, and then expressed as fold change over unsupplemented control.
In order to investigate the effect of growth factors on pPIC proliferation, wells were serum starved for 6 h and then switched to basal media (control), or supplemented with IGF-1 (100 ng/ml; Peprotech), HGF (100 ng/ml; Peprotech), NRG-1 (100 ng/ml; R&D Systems, Minneapolis, Minnesota), or TGF-β1 (5 ng/ml; Peprotech). Each well was supplemented with BrdU (1 μg/ml) every 8 h, fixed after 24 h, and BrdU incorporation was assessed using the BrdU detection kit (Roche) (n = 3/growth factor). Nuclei were counterstained with DAPI. Cells were evaluated using an ApoTome fluorescent microscope (Carl Zeiss). The percentage of BrdUpos cells relative to the total number of cells was determined by counting 5 random fields at ×20 magnification for each dish, and then expressed as fold change over unsupplemented control.
5
Myogenic Differentiation Induction by Growth Factors
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To assess the effect of different growth factors in the induction of myogenic differentiation in vitro, 1 × 104 pPICs were plated in 24-well plates on gelatin-coated coverslips in basal media (control). Individual growth factors (concentrations stated earlier in the text) were supplemented (n = 3 wells/growth factor). Three wells acted as controls, with no growth factors added to the medium. Cells were fixed in 4% paraformaldehyde after 5 days, and myogenic differentiation was quantified by staining for MHC (Developmental Studies Hybridoma Bank). Nuclei were counterstained with DAPI. Cells were evaluated using an ApoTome fluorescent microscope (Carl Zeiss), 5 random fields at ×20 magnification were quantified for each well, and the fusion index was calculated by counting the number of nuclei inside myotubes per total nuclei.
6
Wound Healing and Collagen Invasion Assays
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These experiments, which employed a wound healing and invasion of collagen type IV, were conducted as described previously (7 ). Briefly, the directed migration studies entailed creation of an established size ‘wound’ in confluent monolayer cultures. Images were obtained using an Apotome Fluorescent microscope (Carl Zeiss) and AxioVision software (Carl Zeiss). Quantitative image analysis of wound closure was determined via ImagePro software (Media Cybernertics). Twenty-four hour sera-starved cells were seeded onto type IV collagen-coated microporous polyester membrane (InnoCyte cell invasion kit, Calbiochem, San Diego, CA) and conditioned medium from the OSCC JSCC-3 cell line (previously determined to be highly chemoattractant to OSCC cells) was placed in the lower well. After 16 h of invasion, cells were formalin fixed, stained with crystal violet and cells in upper chamber removed. ImagePro software was used to quantify the invaded cells.
7
Nanobody Binding Assay in A1847 Cells
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A1847 cells (1.5x104 cells/well) were grown on a glass coverslip immerged in 24 well plates for 2 days at 37°C. After washing the cells with PBS, a saturation step was carried on in PBS/BSA 3% for 1 h at 4°C. Then the cells were incubated for 1h at 4°C or 37°C with HA-His-tagged nanobodies (500 nM). The coverslips were washed with PBS-BSA1%, fixed with 4% p-formaldehyde for 30 min at RT, and permeabilised in PBS/0.5% Triton-X100 for 10 min before a 1 h-incubation with AlexaFluor 488-conjugated anti-HA antibody (1/200, LifeTechnologies) at RT. After several washes, the nuclei were stained with DAPI (1/2000 ThermoFischer) for 5 min. Fluorescence was evaluated using an Apotome fluorescent microscope (Zeiss), magnification: x63.
8
Immunofluorescence Staining of Frozen Muscle Samples
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Frozen muscle samples were embedded in OCT and frozen in liquid nitrogen-cooled isopentane. The samples were cut perpendicularly via a cryostat (10-μm thickness). Sections were allowed to cool for 5 min at room temperature and subsequently fixed in 4% paraformaldehyde (PFA) for 5 min. Sections were washed with phosphate-buffered saline (PBS) and incubated with glycine solution to quench the PFA signal. After washing, sections were permeabilized in 0.25% Triton-X for 10 min and washed again. Sections were then incubated in blocking buffer (5% normal goat serum, 2% bovine serum albumin, and mouse-on-mouse blocking reagent-diluted PBS) for 1 h at room temperature. Sections were rinsed in PBS and incubated in primary antibody overnight at 4 °C. The following antibodies were used: laminin (1:200, rabbit, Abcam), anti-MPO (1:200, rabbit, Abcam), and anti-NF-κB p65 (1:200, mouse, Cell Signaling). Sections were rinsed with PBS and incubated in secondary antibody solution diluted in blocking buffer: goat anti-rabbit Alexa Fluor 488 (1:1000) and goat anti-mouse IgG1 Alexa Fluor 546 (1:1000). Sections were rinsed and mounted with fluorescent mounting media containing DAPI (Vector Laboratories). Samples were imaged with a Zeiss Apotome fluorescent microscope.
9
Immunofluorescence and Histological Analysis of Lung Tissue
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For immunofluorescence staining, frozen lung sections were fixed in 3.7% formaldehyde, followed by blocking with 1% BSA in PBS, and stained with a chicken anti-GFP primary antibody (Abcam, Cambridge, MA) and an Alexa Fluor 488 conjugated goat anti-chicken secondary antibody (Life technology, Carlsbad, CA). Images were collected on a Zeiss ApoTome fluorescent microscope using AxioVision software. For hematoxylin and eosin (H&E) staining or Masson’s trichrome staining, whole lungs were inflated and fixed with 10% buffered formalin, dehydrated by ethanol and embedded in paraffin. Sections were cut at a thickness of 3 µm, mounted on glass slides and stained using H&E or Masson’s trichrome. Images were taken on a Olympus BX-51 microscope by a DP-70 camera.
10
KSHV Infection of Lymphatic Endothelial Cells
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