Immunostaining of the frozen kidney sections from the rats was performed using rabbit polyclonal anti-IL-1β (ab9722) and anti-TNF-α (ab9755) antibodies (Abcam, Cambridge, UK). The primary antibody was detected using Alexa Fluor® 594 donkey and anti-rabbit lgG (H+L) labeled secondary antibodies (Cat. no. 1256153; Life Technologies, Grand Island, NY, USA), and incubated for 120 min at room temperature in the dark. DAPI (Sigma-Aldrich) was used for nuclear staining. Images were captured using Nikon DS-Qi1Mc camera attached to Nikon Eclipse 90i fluorescence microscope using an oil immersion objective 60/1.4 NA by Nikon NIS elements AR version 3.2 software.
Nis elements ar version 3
Manufactured by Nikon
Sourced in United States, Japan
NIS Elements AR version 3.0 is a software application developed by Nikon for advanced research microscopy. It provides a comprehensive suite of tools for image acquisition, analysis, and processing. The software supports a wide range of Nikon microscope models and accessories, enabling researchers to capture, manage, and interpret high-quality microscopic images.
Automatically generated - may contain errors
Lab products found in correlation
Ds qi1mc, by Nikon (2 mentions)
Eclipse 90i, by Nikon (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab9755, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab9722, by Abcam (1 mentions)
Eclipse te2000 u inverted microscope, by Nikon (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Anti α smooth muscle actin, by Santa Cruz Biotechnology (1 mentions)
Nis elements software, by Nikon (1 mentions)
Picrosirius red psr, by Thermo Fisher Scientific (1 mentions)
Aperio eslide manager, by Leica (1 mentions)
Eclipse te2000 s inverted fluorescence microscope, by Nikon (1 mentions)
8 well chamber slide, by BD (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
Cell based cholesterol assay kit, by Abcam (1 mentions)
Immunoselect antifading mounting medium, by Dianova (1 mentions)
X cite 120q excitation light source, by Excelitas (1 mentions)
Plan apo, by Nikon (1 mentions)
Eclipse 80i upright microscope, by Nikon (1 mentions)
13 protocols using nis elements ar version 3
1
Immunostaining of Rat Kidney Sections
2
EdU-Based Proliferation Assay for IECs
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The EdU-based staining was performed on 2D-cultured primary IECs. Cells were subcultured from 48-well plates to 96-well plates at a ratio of 1:3, expanded in EM for 4 days, followed by either an additional 2 days in EM, with or without 10 ng/mL BMP9 alone or in combination with 100 ng/mL ALK1–Fc chimera protein, or an additional 2 days in DM. Cells were incubated with 10 mmol/K EdU (10540; Lumiprobe, Hunt Valley, MD) for 3 hours at 37°C. DNA-incorporated EdU was detected by sulfo-cyanine5-azide (A3330; Lumiprobe) through a copper-catalyzed covalent reaction between an azide and an alkyne.49 (link) Images were captured using a Nikon Eclipse TE2000-U inverted microscope and processed using NIS-Elements AR version 3.2 software (Nikon, Tokyo, Japan). The EdU fluorescence area was measured by ImageJ software (National Institutes of Health, Bethesda, MD) and normalized by the total cell area occupied by the Hoechst 33342 fluorescence (62249; Thermo Fisher Scientific Waltham, MA), as we described previously.45 (link)
3
Histological Assessment of Renal Injury
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Formalin fixed renal tissue was sectioned (5 μm) and stained with Periodic Acid-Schiff (PAS) and Picrosirius Red (PSR) for histological examination. Tubular injury was determined in PAS stained tissue sections at magnification of 200× using image analyzing software by NIS Elements AR version 3.0 (Nikon instruments Inc., Melville, NY, USA). Histopathological changes were scored as published earlier [44 (link)]. PSR stained tissue sections were used to study renal interstitial fibrosis by measuring the areas positive for collagen using software by NIS Elements AR version 3.0. The collagen positive renal section areas were expressed as the percentage area fraction relative to the total area analyzed. To minimize observer bias, the tubular injury assessment (cast area calculation) and interstitial fibrosis (collagen positive area calculation) were performed by two observers in a masked fashion without knowledge of the treatment groups.
4
Quantification of Renal Fibrosis by α-SMA
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Kidneys were formalin-fixed and paraffin-embedded slices deparaffinized, re-hydrated, and immunostained with anti-α smooth muscle actin (SMA) antibody (1:100; Santa Cruz Biotechnology, Dallas, TX, USA). Biotinylated rat anti-mouse secondary antibody (1:200) was used with avidin-biotinylated HRP complex (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA, USA) and hemotoxylin used as a counterstain. Slides were then mounted and visualized by light microscopy at 400× magnification. Digital images were obtained for analysis using Nikon NIS Elements Software (Nikon Instruments Inc., Melville, NY, USA). Kidney section area positive for α-SMA was calculated by two experienced blinded reviewers using image analysis software by NIS Elements AR version 3.0 (Nikon instruments Inc.). In different experimental groups, the α-SMA positive renal section areas were expressed as the percentage area fraction relative to the total area analyzed.
5
Quantifying Renal Fibrosis via Picrosirius Red
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Formalin-fixed kidney samples from each animal were paraffin embedded. Kidney samples were sectioned (5μm thickness), mounted on slides, and stained with Picrosirius Red (PSR) (Alfa Aesar, Tewksbury, MA). The stained slides were examined for interstitial fibrosis (PSR-collagen positive area) in the kidney at 200x magnification using NIS Elements AR version 3.0 imaging software (Nikon instruments Inc., Melville, NY, USA). Collagen-positive renal fibrotic area is presented as a percentage area-fraction relative to the total area. Histological analysis and the scoring of collagen-positive kidney area were performed by two observers in a blinded fashion.
6
Histological Evaluation of Renal Fibrosis
7
Histological Assessment of Kidney Fibrosis
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Kidney tissues were fixed in 10% buffered formalin, sectioned (5 μm) and were stained with Picrosirius red (PSR), hematoxylin and eosin (HE) or Periodic Acid-Schiff (PAS) (Alfa Aesar, Tewksbury, MA, USA) for histological examination using ×200 magnification and NIS Elements AR version 3.0 (Nikon Instruments Inc., Melville, NY, USA) imaging software. Kidney fibrosis was determined in PSR-stained sections and data presented as kidney area positive for collagen. The percentage area positive for interstitial collagen was calculated from the mean of 20 cortical and 10 medullary fields for each animal. The percentage area positive for proteinaceous cast was calculated from the mean of eight cortical and five medullary fields for each animal. Glomerular tuft area was calculated from PAS-stained slides. Twenty glomeruli were measured from each kidney sample by two blinded observers, and the average glomerular tuft area was determined. Glomeruli per kidney cortical area were determined in each experimental group. The number of glomeruli is expressed as the average number of glomeruli calculated from ten 10 mm2 fields from the kidney sample of each mouse using Aperio eSlide Manager (Leica Biosystems, Deer Park, IL, USA) in a blinded fashion by two observers.
8
Histopathological Renal Tissue Analysis
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A histopathological study was conducted after fixation of the kidneys with 10% buffered formalin. Renal tissues were sectioned and stained with periodic acid-Schiff (PAS) for histological examination. Histological injury was determined in stained tissue sections at magnification of ×200 using image analysis software by NIS Elements AR version 3.0 (Nikon instruments Inc., Melville, NY, USA). To minimize observer bias, histopathological evaluations were conducted by two observers in a masked fashion without knowledge of the treatment group from which the tissues originated.
9
Multimodal Imaging of Cellular Pathways
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Standard immunofluorescent stainings were performed as described previously [87 (link)] using 8-well-chamber slides (Falcon, Corning, Amsterdam, NY, USA) and Alexa Fluor®-coupled secondary IgG antibodies (Thermo Fisher).
To monitor the autophagic flux using stable transfectants of ptf-LC3 [53 (link)] the cells were treated as indicated and hereafter fixated using 4% paraformaldehyde (PFA, Santa Cruz) for 20 min at RT. After three washing steps with cold PBS the slides were briefly rinsed with de-salted H2O and mounted DAPI containing immunoselect antifading mounting medium (Dianova, Hamburg, Germany).
Cellular cholesterol was stained using the cell-based cholesterol assay kit (Abcam Biochemicals) including filipin III based on the manufacturer’s instructions. Afterwards the slides were prepared for standard immunofluorescent stainng against LAMP1 as described above.
To monitor leaky lysosomes stable transfectants of pmCherry-Gal3, excitation/emission max 586/610 nm were treated as indicated and further processed for standard immunofluorescence staining as described above.
All images were acquired using a Nikon eclipse TE2000-S inverted fluorescence microscope operated by NIS Elements AR version 3.2 (both Nikon, Tokyo, Japan).
To monitor the autophagic flux using stable transfectants of ptf-LC3 [53 (link)] the cells were treated as indicated and hereafter fixated using 4% paraformaldehyde (PFA, Santa Cruz) for 20 min at RT. After three washing steps with cold PBS the slides were briefly rinsed with de-salted H2O and mounted DAPI containing immunoselect antifading mounting medium (Dianova, Hamburg, Germany).
Cellular cholesterol was stained using the cell-based cholesterol assay kit (Abcam Biochemicals) including filipin III based on the manufacturer’s instructions. Afterwards the slides were prepared for standard immunofluorescent stainng against LAMP1 as described above.
To monitor leaky lysosomes stable transfectants of pmCherry-Gal3, excitation/emission max 586/610 nm were treated as indicated and further processed for standard immunofluorescence staining as described above.
All images were acquired using a Nikon eclipse TE2000-S inverted fluorescence microscope operated by NIS Elements AR version 3.2 (both Nikon, Tokyo, Japan).
10
Imaging of Fluorescently Labeled Cells
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Microscopy was performed on coverslips containing fluorescently labeled N2a cells or primary neurons. In our experiments, two different epifluorescence microscopes were used: 1. Olympus IX70 microscope equipped with a 405, 488, 568, and 647 nm channel, X-Cite 120Q excitation light source (Excelitas Technologies), a C11440 ORCA-Flash4-oIT digital camera, and 40x NA 1.3 and 60x NA 1.4 oil immersion objectives. An additional 1.5 magnification of the 60x objective was used when taking the images. 2. Nikon Eclipse 80i upright microscope (RRID:SCR_015572) equipped with a 10x (Nikon plan apo, NA 0.45), 20x (Nikon plan apo, NA 0.75), 40x (Nikon plan apo, NA 1.0, oil immersion), and 60x objectives (Nikon apo VC, NA 1.40, oil immersion). The Nikon microscope was connected to a computer containing Nikon Instruments Software-Elements Advance Research (NIS-Elements AR) version 3.2. The focus of the images was set based on the signal in the ApoE channel.
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