Paraffin embedded kidney sections were processed in batch for immunofluorescence staining using the antibodies EMCN and PDPD. Immunofluorescent images were captured on a Ti2 Widefield microscope (Nikon, Tokyo, Japan) at 20x magnification using standardized imaging parameters for each sample group. The images were then processed using the Vessel Analysis plugin for Fiji software (http://imagej.net/Vessel_Analysis ). For each preprocessed image, 2–3 regions in the kidney cortex were manually encircled to analyze kidney peritubular capillary and lymphatic density while excluding any glomeruli. The vascular density was measured as a ratio of vasculature area to total area of the encircled kidney cortex surface.
Ti2 widefield microscope
Manufactured by Nikon
Sourced in United States, Japan
The Ti2 widefield microscope is a high-performance laboratory equipment designed for advanced imaging applications. It features a large field of view and high-resolution imaging capabilities, making it suitable for a variety of scientific and research purposes. The core function of the Ti2 is to provide users with a powerful and versatile tool for their microscopy needs.
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Lab products found in correlation
Spectra x led light engine, by Lumencor (3 mentions)
Orca 4.0 v2 scmos camera, by Hamamatsu Photonics (3 mentions)
Elements general analysis, by Nikon (2 mentions)
Ds fi3, by Nikon (2 mentions)
Sars cov 2 nucleocapsid, by Thermo Fisher Scientific (2 mentions)
1.4na oil immersion objective lens, by Nikon (2 mentions)
Lu n4 four laser unit, by Nikon (2 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
Mab369, by Merck Group (1 mentions)
A10262, by Thermo Fisher Scientific (1 mentions)
T2928, by Novus Biologicals (1 mentions)
Crest spinning disk confocal microscope, by Nikon (1 mentions)
Labtek plate, by Thermo Fisher Scientific (1 mentions)
Nunclon sphera 96 well plates, by Thermo Fisher Scientific (1 mentions)
Prism 8, by GraphPad (1 mentions)
Nf κb p65, by Abcam (1 mentions)
384 well plate, by Greiner (1 mentions)
F4 80, by Abcam (1 mentions)
Propranolol p0884, by Merck Group (1 mentions)
Cc 3202, by Lonza (1 mentions)
22 protocols using ti2 widefield microscope
1
Kidney Peritubular Capillary and Lymphatic Density
2
Immunocytochemistry Protocol for Synapsin and TH
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Immunocytochemistry was performed following previously published procedures.26 (link),27 (link) The following antibodies were used for immunofluorescence: chicken anti-GFP (Thermo Fisher, A-10262, 1:1000), rabbit ant-GFP (Thermo Fisher, A-11122, 1:1000). Guinea pig anti-synapsin 1/2 (Synaptic System, 106004, 1:500), mouse anti-TH (Sigma, T2928, 1:1000), rabbit anti-TH (Novus Biologicals, NB300-109, 1:1000), rat anti-DAT (Millipore MAB369 1:1000), Immunofluorescence was analyzed using a Nikon Ti-2 wide-field microscope or a Nikon CREST spinning disk confocal microscope. The same imaging parameters were set for each batch of culture. Image stacks were taken at different focal planes at 0.9 μm interval to include the whole cell and a maximum projection image was generated for each stack via ImageJ for analysis. All analyses were done manually. Synapsin 1/2-positive boutons were determined based on the bright punctate staining pattern.
3
Immunostaining of Fungal Hyphae and Fly Hemolymph
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The immunostaining protocol was adapted from Luo et al. (29 (link)). F. oxysporum was grown in 5 ml malt extract broth from a starting concentration of 2.9 × 105 spores/ml. After overnight shaking at room temperature, fungal hyphae were collected by centrifugation at 1,000 g for 10 min and resuspended in PBS. Hemolymph was collected via the Zymo-Spin IC column method (23 (link)) from 420 male flies that had been induced with heat-killed M. luteus 24 h prior and incubated at 29°C, yielding ~35 μl cell-free hemolymph. Next, aliquots of 200 μl hyphae and 35 μl hemolymph were shaken at room temperature for 30 min. The samples were washed three times with PBS before fixation with 4% formaldehyde for 1 h. After washing another three times with PBS, samples were blocked for 1 h with 5% BSA. Samples were then incubated with α-FLAG antibody (1:200) overnight at 4°C. After washing with PBS, samples were stained for 2 h with donkey α-mouse Alexa555 (1:400) and DAPI (1:200) and then washed and mounted on slides. Samples were imaged with a Ti2 Widefield microscope (Nikon) and analyzed with the NIS- elements software and OMERO.
4
3D Spheroid Growth Dynamics
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Spheroids were generated as described by Gilbert-Ross et al. [81 (link)]. Briefly, cells were grown in Nunclon Sphera 96-well plates (Thermo-Fisher Scientific) at a concentration of 3000 cells per well. After 3 days in culture, cells were transferred using a wide-bore pipette tip to 2 mg/mL collagen (Corning) in 4-well LabTek plates (Nunc). collagen was allowed to gel at 37 °C for 1 h; then, complete media was added to the spheroids. Gels were imaged using a Ti2 widefield microscope (Nikon) at 0, 24, and 48 h. Spheroid area was quantified using Fiji software. Reported spheroid area values are normalized to 0-h spheroid area of the same spheroid.
5
Cortex Region Imaging and Analysis
6
NF-κB p65 Translocation Assay in Macrophages
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The NF-κB p65 translocation assay was performed by seeding primary peritoneal macrophages at 4000 cells/well in a 384-well plate (Greiner Bio-One, Kremsmünster, Austria). They were kept in complete media (DMEM + 10% FBS and gentamycin) for overnight incubation. The next day, the media was replaced with serum-free media containing the following conditions: inflammatory-only (IFN-γ + LPS), growth factors (VEGF at 50 ng/mL, PDGF at 50 ng/mL, combined VEGF + PDGF at 50 ng/mL each), inflammatory with growth factors (IFN-γ + LPS + VEGF, IFN-γ + LPS + PDGF, IFN-γ + LPS + VEGF + PDGF) and an untreated control. They were fixed using 4% paraformaldehyde (PFA) at 3, 24 and 48 h. Further, they were stained using NF-κB p65 (1:500, Abcam), F4/80 (Abcam) and Hoechst. Images were acquired using a Nikon Ti2 widefield microscope (Tokyo, Japan) at 20× objective. They were processed using CellProfiler 3.1.9 software (Broad Institute, MIT, Cambridge, MA, USA) to assess nuclear: cytoplasm NF-κB p65 ratio.
7
Imaging ICAM-2 Expression in Laser-Induced CNV
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Eyes were treated as previously described6 (link),11 . Briefly, mice were sacrificed 2 weeks after laser-induced CNV. Enucleated eyes were fixed for 1 h in 1% paraformaldehyde (#15,713-S, Electron Microscopy Sciences, Hatfield, PA) at room temperature. Eyes were washed in Tris-buffered saline (TBS) and dissected to remove conjunctiva, cornea, iris, ciliary body, lens, and retina leaving a posterior eye cup of RPE, choroid, and sclera. Eye cups were blocked in TBS + 5% Donkey serum (S30, Sigma-Aldrich), then treated with an anti-ICAM-2 primary antibody (1:500, Table 1 ), and Alexa Fluor 488-conjugated anti-rat secondary antibody (Table 1 ). Pictures were captured on a Ti2 widefield microscope (Nikon, Melville, NY). Area was quantified using Fiji by a reviewer blinded to animal identification.
8
Choroidal Angiogenesis Assay Protocol
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Eyes were carefully enucleated and placed in ice-cold PBS. Eyes were dissected in EGM2-MV supplemented (hydrocortisone was omitted) medium (CC3202; Lonza, Walkersville, MD, USA) into posterior eye cups containing sclera, RPE, and choroid complex. The peripheral choroid was separated from the central choroid, cut into 0.5 mm × 0.5 mm pieces, and placed into growth factor reduced Matrigel (#356231; Corning, Bedford, MA, USA) in a 48-well plate on ice. The Matrigel was solidified by incubating at 37°C for 10 minutes. EGM2-MV medium was changed every 2 days. Sunitinib (PZ0012) and propranolol (P0884) were purchased from Sigma (St. Louis, MO, USA). Sunitinib was added on Day 0, and propranolol was added on either Day 0 or Day 2. Pictures were taken on a Nikon Ti2 Widefield microscope (Buffalo Grove, IL, USA) using a 4× objective and Nikon NIS Elements software. Images were analyzed with the Nikon Elements General Analysis. Images were preprocessed with edge detection and segmented with thresholding. The largest area was measured for each image. The central choroidal tissue area was subtracted from the total angiogenesis area.
9
Widefield Imaging of Primary Hippocampal Neurons
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Primary hippocampal neurons were imaged using a custom widefield inverted Nikon Ti-2 widefield microscope equipped with CFI PLAN APO LAMBDA 60× 1.4 NA oil immersion objective lens (Nikon), Spectra X LED light engine with a C-FL DAPI SOLA, C-FL GFP/FITC/Cy2, C-FL DSRed/TRITC/Cy3, and C-FL Cy5 filter sets (Semrock, Rochester, NY, USA) and a custom Cy7 Penta Pass Filter for NIR SpectraX, DAPI-FITC-TRITC-CY5-CY7 bandpass filter, and an Orca-Fusion sCMOS camera (Hamamatsu, Hamamatsu, Japan) with a pixel size of 6.5 μm and a peak quantum efficiency of 80%. The x-y pixel size is 107.5 nm, and the z-step size is 200 nm. A stack of 41 optical planes (0.2 μm step) was acquired consecutively in 5 channels (652 nm, 573 nm, 759 nm, 495 nm, 409 nm) using Nikon Elements software (Nikon Corporation, Japan).
10
Choroid Angiogenesis Assay in Mice
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Experiments were performed as described [29 (link)] on female mice. Pieces of choroid were dissected and placed on Matrigel (Corning) in endothelial cell growth media (EGM2, Lonza). Media were changed every 2 days and pictures were taken on day 4 with a Nikon Ti2 Widefield microscope using a 4 × objective and Nikon NIS Elements software. Images were analyzed with Nikon Elements General Analysis. Images were preprocessed with edge detection and segmented with thresholding. Total area was measured for each image with central choroidal tissue subtracted from the angiogenesis area.
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