Cells were grown on coverslips and transfected according to protocols provided by manufacturers with noted plasmids. After 24 h, cells were fixed using ice-cold 4% PFA for 10 min. Cells were then washed three times with PBS and blocked with blocking buffer (4% BSA + 0.1% Saponin in PBS) at 25°C for 30 min. Cells were incubated with primary antibodies at 4°C overnight, washed three times with PBS buffer, and then incubated with appropriate secondary antibodies for 1 h at 25°C. Slides were examined under a laser scanning confocal microscope (Zeiss LSM 880) using 63× (NA 1.4) or 40× (NA 1.3) lenses at RT. For immunofluorescence staining of skeletal muscles sections, mice were perfused with 4% PFA. Skeletal muscles were dissected and fixed in 4% PFA overnight, and then incubated in 15% sucrose in PBS for 4 h or overnight followed by an incubation of 30% sucrose in PBS overnight before the frozen sections were prepared. After freezing the sections, the skeletal muscles sections were stained with primary antibodies and the appropriate secondary antibodies. Sections were mounted and imaged using a fluorescent microscope (Zeiss Axioplan2) under a 63× (NA 1.4) lens at RT. For GFP-LC3 imaging, the sections were directly mounted, and images were taken using a fluorescent microscope (Zeiss Axioplan2) under a 63× (NA 1.4) lens at RT.
Axioplan 2
Manufactured by Zeiss
Sourced in Germany, United States, United Kingdom, Switzerland, Canada, Japan, France, Austria, Colombia
The Axioplan 2 is a microscope system designed for advanced optical analysis. It features a modular design that allows for the integration of various accessories and components to suit a wide range of applications. The core function of the Axioplan 2 is to provide high-quality imaging and analysis capabilities for scientific and research purposes.
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Lab products found in correlation
Axiovision software, by Zeiss (24 mentions)
Axiovision 4, by Zeiss (23 mentions)
Axiocam mrm, by Zeiss (21 mentions)
Axiocam, by Zeiss (21 mentions)
Lsm 510 meta, by Zeiss (17 mentions)
Lsm 510, by Zeiss (16 mentions)
Axiocam hrc, by Zeiss (14 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (13 mentions)
Axiovision, by Zeiss (12 mentions)
Lsm 880, by Zeiss (12 mentions)
Vectashield, by Vector Laboratories (9 mentions)
Lsm 710, by Zeiss (9 mentions)
Axiocam mrc5, by Zeiss (9 mentions)
Paraformaldehyde (pfa), by Merck Group (8 mentions)
Lsm 780, by Zeiss (8 mentions)
C4742 95, by Hamamatsu Photonics (7 mentions)
Axiocam hrm, by Zeiss (7 mentions)
Openlab software, by PerkinElmer (7 mentions)
Plan apochromat, by Zeiss (6 mentions)
Lsm 700, by Zeiss (6 mentions)
859 protocols using axioplan 2
1
Immunofluorescence Staining and Imaging
2
Quantifying Microglia CD68 and PdgfRα+ in Hypothalamus
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For CD68 measurements, individual microglia cells were isolated from the ventricle and periphery areas in the rostral tuberal hypothalamus at E15.5 from images taken using a × 20 objective on a Zeiss Axioplan 2 manual compound microscope. Microglia denoted as being located in the ventricular area were within the 10% distance bin of relative distance to the ventricle, and microglia denoted as being located within the periphery were located between the 30 and 90% distance bins of relative distance to the ventricle. The area of CD68 and Iba1 immunostaining was calculated for each image using the measurement tool in ImageJ. CD68 total area was calculated using the area of CD68 within each individual microglia. The CD68 load was calculated by dividing the area of CD68 by the area of Iba1 resulting in a percentage of the area containing CD68 for each individual microglia. A Student’s t test was used to determine statistical significance between groups. For PdgfRα+ area coverage calculations, uniform sized images were taken from randomly chosen white matter and grey matter areas in the rostral tuberal hypothalamus at E15.5 and E17.5 using a × 40 objective on a Zeiss Axioplan 2 manual compound microscope. Total area of PdgfRα+ coverage was calculated per image. An ANOVA with Tukey’s post hoc was used to determine statistical significance between treatments and embryonic stages.
3
Histological Analysis of Hepatic Tissue
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Hepatic tissue samples were fixed directly in 10% neutral buffered formalin solution and embedded in paraffin wax. Paraffin blocks were cut to sections of 5 μm using a microtome (HM325, Thermo Fisher Scientific, Waltham, MA, USA). Hematoxylin-eosin (H&E) staining was performed according to the manufacturer’s guidelines (Merck Millipore, Darmstadt, Germany). Digital images of tissue slices were captured using a Zeiss microscope Mod. Axioplan 2 (Zeiss, Jena, Germany).
Oil Red O staining was performed on frozen liver sections (5 µm) previously fixed in 10% formalin for 5 min as previously described [60 (link)]. Briefly, slides were rinsed three times with absolute propylene glycol and then placed in 0.5% Oil Red O stain solution in propylene glycol for 30 min before being rinsed with 85% propylene glycol for 1 min and counterstained with hematoxylin. Thereafter, the slides were washed with distilled water and mounted with aqueous mounting medium (Sigma, St. Louis, MO, USA). Sections were observed with a Zeiss microscope Mod. Axioplan 2 (Zeiss, Jena, Germany).
Oil Red O staining was performed on frozen liver sections (5 µm) previously fixed in 10% formalin for 5 min as previously described [60 (link)]. Briefly, slides were rinsed three times with absolute propylene glycol and then placed in 0.5% Oil Red O stain solution in propylene glycol for 30 min before being rinsed with 85% propylene glycol for 1 min and counterstained with hematoxylin. Thereafter, the slides were washed with distilled water and mounted with aqueous mounting medium (Sigma, St. Louis, MO, USA). Sections were observed with a Zeiss microscope Mod. Axioplan 2 (Zeiss, Jena, Germany).
4
Spectral Karyotyping and CDKN2A FISH Analysis
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Spectral karyotyping was performed according to the manufacturer’s protocol using 24-color human SKY paint probes (Applied Spectral Imaging, CA). Fluorescent in Situ Hybridization (FISH) analysis was carried out using a CDKN2A probe (Abbott Molecular, IL) encompassing the overlapping genes encoding p16INK4a and p14ARF. Spectral images of the hybridized metaphases were acquired using a SD301 SpectraCubeTM system (Applied Spectral Imaging Inc., CA) mounted on top of an epi-fluorescence microscope Axioplan 2 (Zeiss). Images were analyzed using Spectral Imaging 6.0 acquisition software (Applied Spectral Imaging Inc., CA). At least 10 SKY hybridized metaphases were analyzed in this experiment. FISH analyses were performed under an Axioplan 2 (Zeiss) fluorescence microscope coupled with a CCD camera (ASI) and images were captured with FISH view 5.5 software (Applied Spectral Imaging Inc., Vista, CA).
5
Quantifying Citrullinated Histone H3 in Blood and Brain
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Isolated blood PMNs were cytocentrifuged (ThermoFisher Scientific, Waltham, MA, USA) at 500 rpm for 8 min at RT and then fixed with 4% paraformaldehyde (PFA, Sigma Aldrich, St. Louis, MO, USA). Cytospin slides were then blocked with 1% normal goat serum for 30 min and incubated overnight with anti-CitH3 antibody (ab18956-100; Abcam, Cambridge, UK) at 4 °C. DNA was stained with DAPI (4′,6-diamidino-2-phenylindole; Sigma Aldrich, St. Louis, MO, USA) to confirm the nuclear localization of CitH3 and nuclei. Slides were observed under a fluorescence microscope (Axioplan 2; Zeiss, Oberkochen, Germany). To prepare brain tissue, animals were sacrificed 24 h after surgery and brains were isolated and fixed with 4% paraformaldehyde (PFA; Sigma Aldrich, St. Louis, MO, USA) by transcardiac perfusion and then stored in the same solution overnight at 4 °C. Brain sections (40 μm) were prepared using a vibratome. Primary antibodies for anti-CitH3 (ab18956-100; Abcam, Cambridge, UK) and anti-RECA (MCA970; Bio-Rad, Hercules, CA, USA) were diluted 1:200. Brain sections were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Sigma Aldrich, St. Louis, MO, USA) to visualize nuclei, and observed under a fluorescence microscope (Axioplan 2; Zeiss, Oberkochen, Germany). The numbers of CitH3-positive cells in 0.16 mm2 (0.4 × 0.4 mm) were scored.
6
Wound Tissue Histological Analysis
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Wound samples containing both wound tissue and normal undamaged skin tissue were collected using a scalpel. Tissue samples were then directly placed in neutral buffered formalin (Sigma) and fixed overnight at 4 °C. After serial dehydration, the samples were embedded in paraffin blocks, sectioned using a microtome (Leica), and routine hematoxylin and eosin (H&E) staining was performed. Stained tissue sections were then imaged with bright field microscopy (Axioplan 2, Zeiss). Tissue sections from the same samples were also stained with 4′,6-diamidino-2-phenylindole (DAPI) and visualized and imaged using fluorescence microscopy (Axioplan 2, Carl-Zeiss, Oberkochen, Germany). For both H&E- and DAPI-stained sections, 174 images were taken of the sections, which were then stitched together to gain a panoramic view using Axioplan 2. Image J was used to determine the diameter of the wound.
7
Analyzing Adipocyte Morphology
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Microscopy was performed on a Nikon ECLIPSE TE2000-U with NIS-Elements F software (Nikon), Zeiss AxioPlan 2, and Zeiss AxioPlan 2 IE microscopes. Adipocytes were traced and their area measured using ImageJ software.
8
Fluorescence Microscopy Imaging Protocol
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Microscopy was performed using Zeiss Axioplan 2 and Axioplan 2 IE MOT epifluorescence microscopes and an Olympus FV1000 confocal microscope. Epifluorescence images were acquired on the Axioplan 2 unless specifically noted below. The Axioplan 2 is equipped with a Zeiss 50-W HBO AC mercury lamp source and uses the following filters: eGFP, excitation (ex.) 470 ± 20 nm, emission (em.) 540 ± 20 nm; eCFP, ex. 436 ± 10 nm, em. 480 ± 20 nm; eYFP, ex. 500 ± 10 nm, em. 535 ± 15 nm; and mRFP or mCHERRY, ex. 546 ± 6 nm, em. 650 ± 37.5 nm. The Axioplan 2 IE MOT used 100-W FluoArc or an X-Cite 120 (EXFO Life Sciences) mercury lamp source and was equipped with the following filters: GFP, ex. 480 ± 20 nm, em. 510 ± 10 nm (Fig. 4, MGG_09470 and MGG_10914, direct fusions) and mCHERRY, ex. 535 ± 35 nm, em. 610 ± 32 nm (Fig. 5D, right-hand panel). Imaging on the FV1000 confocal microscope used a PLAPO ×40 WLSM objective. Excitation and emission wavelengths were 488 and 510 nm, respectively, for eGFP and 543 and 581 nm, respectively, for mRFP.
9
Histological Analysis of Tibia Growth
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At the end of the culture period, E15.5 tibiae were selected from control media and from treatments that had the greatest effect on longitudinal growth (1000 µM ouabain, 0.1 µM monensin), fixed in 10% NBF and decalcified using Cal-Ex IITM (Fisher Chemical). Tissues were then dehydrated, embedded in paraffin, and sectioned in the coronal plane at 4 µm. Sections were deparaffinized in xylene and hydrated. Tibia sections were stained using Wiegert’s Iron Haematoxylin (Sigma), 0.05% Fast-Green (FCF) (Sigma) and 0.1% Safranin-o solution (Sigma). The stained sections were imaged using a digital microscope (Axioplan 2, Zeiss) with attached camera (Optronics), using StereoInvestigator v7 or PictureFrame.
10
Histological Analysis of Calvaria Defects
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The calvaria were separated from the cranium, and the specimens were fixed in 4% paraformaldehyde and then dehydrated via a graded series of ethanol treatments (70–100%). Non-decalcified bone specimens were infiltrated and embedded using Technovit® 9100 (Heraeus Kulzer, Wehrheim, Germany). Blocks were cut into 7 µm slices with a microtome (SM2500, Leica, Frankfurt am Main, Germany) before staining. Only sections in the center of the defects were selected. The sections were stained with HES (hematoxylin, eosin, and safranin) and Goldner’s stain and then examined using a light microscope (Axioplan 2; Zeiss, Darmstadt, Germany).
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