Transwell inserts of 6.5mm (24 well) and 12mm (12 well) with a pore size of 8μm (Corning) were used. 5 × 104 (6.5mm) or 7.5 × 104 (12mm) WT or Cn−/− fibroblasts were plated in inserts containing serum-free media in the upper chamber and 10% serum in the lower chamber. For migration assays, inserts were uncoated; for invasion assays, inserts were coated with 75μl 1mg/ml neutralized type I rat tail collagen for 1 hour before cell seeding. For invasion assays, uncoated transwells were used as positive controls; for migration and invasion assays, serum-free media was placed in the lower chamber as negative controls. 24 hours following plating, the upper chamber was wiped with a cotton swab to remove remaining cells, washed with PBS, fixed and stained with 0.5% crystal violet in 25% methanol solution for 15 minutes, washed in deionized water, and dried before imaging. Images were taken by tile-scanning using a Zeiss Axio Imager M2 upright microscope with Zen Pro software. Crystal violet-positive area was measured using ImageJ. Assays were performed in triplicate and experiments repeated three times.
Axio imager m2 upright microscope
Manufactured by Zeiss
Sourced in Germany
The Axio Imager M2 is an upright microscope designed for advanced research applications. It features a sturdy, ergonomic design and high-quality optics to provide clear, detailed images. The microscope is equipped with a range of illumination options and can accommodate a variety of sample types. Its modular design allows for customization to meet the specific needs of researchers and laboratories.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Axiocam hrm camera, by Zeiss (2 mentions)
Anti iba1 antibody, by Fujifilm (2 mentions)
Z033401 2, by Agilent Technologies (2 mentions)
Dapi fluoromount g, by Southern Biotech (2 mentions)
Axiocam 506 mono, by Zeiss (2 mentions)
Transwell insert, by Corning (1 mentions)
Fv1200 laser scanning confocal microscopy, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Axio observer inverted microscope, by Zeiss (1 mentions)
Smad2, by Cell Signaling Technology (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
Eclipse ti e tirf microscope, by Nikon (1 mentions)
Lsm800 inverted confocal microscope, by Zeiss (1 mentions)
Anti dsred, by Takara Bio (1 mentions)
Cm3050s cryomicrotome, by Leica (1 mentions)
Anti epcam af488, by BioLegend (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
18 protocols using axio imager m2 upright microscope
1
Transwell-Based Migration and Invasion Assay
2
Isolation and Characterization of α4β7+ CLPs
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α4β7+ CLPs (Lin−CD127+c-KitintSca-1intFlt3+α4β7+) isolated by FACS or intestinal frozen sections were fixed with 4% PFA (Sigma-Aldrich) for 20 min at room temperature, perforated with PBS containing 1% Triton X-100 for 20 min, blocked with 5% donkey and 5% rat serum for 1 h at room temperature, incubated with appropriate primary antibodies at 4°C overnight, and then incubated with fluorescence-conjugated secondary antibodies. DAPI was used for nucleus staining. Cells were visualized with an Olympus FV1200 laser scanning confocal microscopy. Intestinal frozen sections were visualized with an AxioImagerM2 upright microscope (Zeiss).
3
Immunofluorescence of Cell-Cell Interactions
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Cells were treated and grown on 48-well plates (conditioned medium experiments) or on glass coverslips in the bottom compartment of 6-well co-culture plates (co-cultures experiments, pore size 0.4 μm) and fixed with 4% paraformaldehyde. Cells were then permeabilized with 0.1% Triton X-100 in PBS for 20 min and blocked with 5% FBS in PBS for 30 min at room temperature (RT). The primary antibodies used were: mouse monoclonal antibodies against β-CATENIN (1:100; BD Biosciences #610153), E-CADHERIN (1:300; BD Biosciences #610181) and rabbit monoclonal antibody SMAD2 (1:100; Cell signaling D43B4). Secondary labeled antibodies were goat antibody against mouse IgG, Alexa Fluor 488 dye-conjugated (1:500; Invitrogen) and goat antibody against rabbit IgG, Alexa Fluor 594 dye-conjugated (1:500; Invitrogen). F-ACTIN was stained with phalloidin-TRITC (1:500; Sigma-Aldrich). DNA was stained with DAPI (1 μg/mL; Sigma-Aldrich). Fluorescent images were taken with the Zeiss Axio Imager M2 upright microscope (for co-culture experiments) or with the Axio Observer inverted microscope (for conditioned medium experiments). Images were processed using Zeiss Zen and ImageJ software.
4
Imaging and Analysis of Phase-Separated Biomolecular Assemblies
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DIC images were captured using a Zeiss AxioImager M2 upright microscope. Raw, unprocessed DIC images are shown in the paper.
Epifluorescence images were captured with a Nikon Eclipse Ti-E TIRF microscope in epifluorescent mode. Raw, unprocessed images were analyzed using the Fiji (ImageJ) software. For illustration purposes, images were background corrected (SI Appendix, Supplementary Materials and Methods ). Methods of determination of size distribution and fluorescent partitioning are described in SI Appendix, Supplementary Materials and Methods .
FRAP experiments were performed at 23 °C using an inverted LSM800 confocal microscope (Zeiss).
For evaluation of droplet volumes, microscopic images were taken on a Zeiss Spinning Disk system at 24 °C. Image analysis methods are described inSI Appendix, Supplementary Materials and Methods .
Epifluorescence images were captured with a Nikon Eclipse Ti-E TIRF microscope in epifluorescent mode. Raw, unprocessed images were analyzed using the Fiji (ImageJ) software. For illustration purposes, images were background corrected (
FRAP experiments were performed at 23 °C using an inverted LSM800 confocal microscope (Zeiss).
For evaluation of droplet volumes, microscopic images were taken on a Zeiss Spinning Disk system at 24 °C. Image analysis methods are described in
5
Immunohistochemical Imaging of ChiaRed Lungs
6
Immunofluorescence of Lung Sections
7
Proximity Ligation Assay for Twist1, Smad2/3/4
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Cells were seeded on chamber slides and fixed with 4% formaldehyde for 10 min at room temperature, washed twice with PBS containing 0.02% Tween 20, and permeabilized with 0.5% Triton X-100/PBS solution (blocking solution) for 30 min at room temperature. Primary antibodies against Twist1 (mouse IgG), Smad2 (rabbit IgG), Smad3 (rabbit IgG), or Smad4 (rabbit IgG) were incubated in blocking solution at 4°C overnight. Assays were then performed with the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich, DUO92101-1KT) according to the manufacturer’s instructions using anti-mouse MINUS and anti-rabbit PLUS proximity ligation assay probes. Images were acquired by a Zeiss Axio Imager M2 upright microscope using a ×40 objective and analyzed for fluorescence per nucleus with HALO software.
8
BrdU Incorporation Assay for PDLSCs
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For BrdU incorporation assay, PDLSCs (1 × 103), including Cont-PDLSCs, PD-PDLSCs, MOCK-PD-PDLSCs, EPO-PD-PDLSCs, siCONT-PDLSCs, and siEPOR-PDLSCs, were cultured on a 2-well chamber slide (Thermo Fisher Scientific) in CGM for 2 days and examined by using a BrdU staining kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The nuclei were lightly stained with hematoxylin. The cell images were captured under an Axio Imager M2 upright microscope (Carl Zeiss Microscopy) and the ratio of BrdU-positive nuclei was quantified using ImageJ software (National Institutes of Health).
9
Bright-field and DIC Microscopy for Imaging
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Bright-field microscopy was performed using a Nikon ECLIPSE E600 microscope equipped with a Keyence VB-7010 charge-coupled device (CCD) color camera system to acquire images with 20× and 40× water immersion lenses or a 100× oil immersion lens (Plan Fluor). Differential interference contrast (DIC) optics and a Zeiss Axio Imager M2 Upright microscope equipped with an AxioCam MRm digital camera were used to acquire images with a 100× oil immersion lens (Plan Apochromat) and Axiovision 4.8 software.
10
Brain Tissue Preparation and Immunostaining
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Animals were euthanized by rapid decapitation two hours after conclusion of the isoflurane exposure. Brains were isolated and preserved in 4% PFA for 16–24 hours at 4°C. A Leica VT1200 Vibratome was used to obtain 50 μm thick coronal sections from Paxinos plate P6 #25 to #34 [21 ], every fifth section was collected for analysis. For immunohistochemistry free-floating sections were blocked and permeabilized with 2% donkey serum in 0.2% Triton X-100/PBS for four hours at room temperature followed by antibody staining with 1:1000 anti-cleaved caspase 3 (Cell Signaling Technology) and subsequently 1:1000 anti-rabbit Alexa 568 (ThermoFisher) and 1:5000 Hoechst 33342 (ThermoFisher). Sections were mounted on PermaFrost Plus slides (ThermoFisher), coated in FluoroMount-G (SouthernBiotech) and coverslipped. For Fluorojade C staining sections were mounted to PermaFrost Plus slides and air dried for 24 hours. If not immediately processed, these slides were stored at -80°C. Fluorojade C staining was carried out as previously described [22 (link)]. Following staining sections were coated in DPX Mounting Media (SigmaAldrich) and coverslipped. Sections were imaged with a Zeiss Axio Imager M2 upright microscope equipped with an ApoTome.2. Immunohistochemical analysis was performed using Fiji (NIH) by an observer blind to genotype and experimental conditions.
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