For the FGF-2 induced angiogenesis zebrafish yolk membrane (ZFYM) [61 (link)], 24 hpf embryos were exposed to 1-phenyl-2-thiourea (PTU) to prevent the pigmentation. At 48 hpf, embryos were manually dechorionated with forceps, anesthetized with tricaine (0.016%), and injected into the perivitelline space with 2 mL FGF-2 (1 mg/mL). The injection was performed in the proximity of developing subintestinal vessels (SIVs) using borosilicate needles and a Picospritzer microinjector (Eppendorf, Hamburg, Germany). After injection, embryos were incubated for 24 h more in the absence or the presence of Solo F–OH. Finally, embryos were fixed in 4% paraformaldehyde (PFA), stained for endogenous alkaline phosphatase (AP) activity, and photographed under a Leica MZ16 F stereomicroscope equipped with a DFC480 digital camera and ICM50 software (Leica, Wetzlar, Germany). Evaluation of the angiogenic response was performed by assigning negative (–, no response to FGF-2 injection), positive (+, mild response), or very positive (++, strong response) scores to the embryos.
Dfc 480 digital camera
Manufactured by Leica camera
Sourced in Germany
The DFC 480 is a digital camera designed for laboratory use. It features a high-resolution imaging sensor and advanced data processing capabilities to capture detailed images for scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mz16f stereomicroscope, by Leica camera (1 mentions)
Photomicroscope, by Leica camera (1 mentions)
Ab45688, by Abcam (1 mentions)
3 3 diaminobenzidine chromogen, by Agilent Technologies (1 mentions)
Ab34710, by Abcam (1 mentions)
A2547, by Merck Group (1 mentions)
Ab6586, by Abcam (1 mentions)
Las leica imaging software, by Leica camera (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Carbon tape, by Ted Pella (1 mentions)
Leica mz16 stereomicroscope, by Leica camera (1 mentions)
Scanning electron microscope, by Hitachi (1 mentions)
Dm2000 microscope, by Leica camera (1 mentions)
Epoxy resin, by Merck Group (1 mentions)
Mz16 stereomicroscope, by Leica camera (1 mentions)
Dmr light microscope, by Leica camera (1 mentions)
Leica photomicroscope, by Leica camera (1 mentions)
Rabbit anti 8 ohdg polyclonal antibody, by Bioss Antibodies (1 mentions)
Dako protein block, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine dab chromogen substrate solution, by Agilent Technologies (1 mentions)
10 protocols using dfc 480 digital camera
1
FGF-2 Induced Angiogenesis in Zebrafish Yolk Membrane
2
Histopathological Analysis of Kidney Injury
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Formalin-fixed hemisected kidneys were embedded in paraffin and stained with Masson’s trichrome or Periodic Acid Schiff (PAS). They were examined using a light microscope (Leica photomicroscope linked to a DFC 480 digital camera) and five non-overlapping fields were captured for each kidney. Tubular interstitial injury was an aggregate measure of tubular dilatation, separation, atrophy and interstitial fibrosis as measured by a blinded independent nephrologist. Images were analyzed using an Olympus microscope (Olympus, Japan) and Image J software.
3
Quantitative Immunohistochemistry of Kidney Fibrosis
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Paraffin-embedded kidney sections (4 micron) were dewaxed in xylene and rehydrated in graded concentrations of ethanol before heat epitope retrieval in 10 mM citrate buffer, pH6. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide. After a 10 min pre-incubation with 10% protein block (Dako, CA) the sections were incubated overnight at 4 °C with primary antibodies against LOXL2 (1:500 dilution, sc-66950, Santa Cruz, USA), fibronectin (dilution 1:1000, ab45688, Abcam, Cambridge, UK), collagen I (dilution 1:500, ab34710, Abcam), collagen IV (dilution 1:500, ab6586, Abcam), αSMA (dilution 1:100, A2547, Sigma Aldrich, USA). The sections were developed with 3,3′-diaminobenzidine chromogen (Dako, CA, USA) after incubation with the respective horseradish-peroxidase (HRP) tagged secondary antibodies (Dako) and then counterstained with haematoxilin. The quantification was performed by capturing 10–12 non-overlapping fields of renal cortex from stained sections at 200× magnification with a Leica microscope linked to a DFC 480 digital camera (Leica, Wetzlar, Germany). The percentage of stained area relative to the whole area in each field was assessed with image J software (Java-based software program, National Institutes of Health).
4
Histopathological Assessment of Kidney Fibrosis
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Paraffin embedded kidneys sections (2 μm) were stained with Masson’s Trichrome and Periodic Acid Schiff (PAS). Assessment of glomerulosclerosis and tubulointerstitial fibrosis was done by two examiners independently. Twenty non-overlapping fields were captured under an Olympus photo light microscope linked to a Leica DFC 480 digital camera. A semiquantitative glomerulosclerosis index (GSI) score was graded on a scale of zero to four (0_normal; 1_involvement of <25% of the glomerulus, 2_ involvement of 25–50% of the glomerulus; 3_involvement of 50–75% of the glomerulus and 4_totally sclerosed glomeruli)41 (link). The whole kidney average GSI score was obtained by averaging scores from all counted glomeruli in one section. For tubulointerstitial scoring ten non-overlapping fields were assessed in a similar grading system to the GS score as previously described42 (link). Paraffin sections of liver, lung and heart from control and control animals treated for 24 weeks with the LOXL2 inhibitor were stained with haematoxylin and eosin (HE) and evaluated for structural damage by an independent pathologist.
5
Visualizing Zebrafish Vascular Development
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Wild-type zebrafish embryos were raised with or without purified snail extracts from H. aspersa. At 26 hpf, 20 embryos without any obvious deformities were selected for each condition and fixed with 4% (v/v) paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MO, USA), dehydrated in 100% (v/v) methanol, and stored at −20 °C. WISH was performed with the vascular probe fli1. The preparation of the probes and WISH in zebrafish embryos was performed as previously described in [33 (link)]. WISH images were taken with a Leica MZ16F stereomicroscope equipped with a DFC 480 digital camera and LAS Leica Imaging software (Leica, Wetzlar, Germany) at 63× magnification.
WISH images were quantified using ImageJ Fiji software, as follows: The region of the embryo tail containing the WISH signal (approximately from the mid-yolk region to the tip of the tail) was selected, and the intensity was measured. An equal area of the tail outside of the stained area was selected to determine the background. The value of the colorimetric signal was then obtained by subtracting the background from the measured intensity.
WISH images were quantified using ImageJ Fiji software, as follows: The region of the embryo tail containing the WISH signal (approximately from the mid-yolk region to the tip of the tail) was selected, and the intensity was measured. An equal area of the tail outside of the stained area was selected to determine the background. The value of the colorimetric signal was then obtained by subtracting the background from the measured intensity.
6
Quantifying Drosophila Morphological Traits
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Fly heads were removed from newly eclosed flies. Heads were passed through a graded alcohol series for 24 hours each (25%–50%–75%-absolute), passed through Hexamethyldisalizane, and mounted on EM stubs using carbon tape (Ted Pella). Fly heads were dried for 24 hours, sputter coated with gold, and examined with a Hitachi scanning electron microscope at an accelerating voltage of 20 kV. Images were acquired as TIFF files that were collated using Adobe Illustrator. For quantitative analysis of eye size (facet number), crosses were established in duplicates, and ≥15 adult flies for each genotype were photographed using a Leica MZ16 stereomicroscope equipped with a Leica DFC-480 digital camera. Facet numbers were manually counted from TIFF images of the eyes and subjected to statistical analysis. For bristle phenotypes, newly eclosed adults were photographed using a Leica MZ16 stereomicroscope or by scanning EM. For quantitative analysis of the bristle phenotypes, multiple crosses were established (triplicates), and adults were scored for bristle defects. Wing margin phenotypes were documented using a Leica MZ16 stereomicroscope. Eye size (facet counts) and bristle and wing defects were statistically analyzed using the Student’s t-test, with the exception of Fig. 1F , which was determined using the chi-squared test.
7
Histological Analysis of Sole Skin and Gut
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Individual skin and gut samples from n = 5–6 pigmented and n = 4–6 pseudo-albino juvenile sole were fixed overnight at 4 °C in 4% paraformaldehyde, washed in PBS and stored in 70% ethanol. Skin samples were decalcified overnight in 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 8 prior to processing. Skin and gut samples were dehydrated in a graded ethanol series (70–100%), saturated in xylene and impregnated with paraffin wax. Serial 5 μm sections were mounted on poly-L-lysine coated glass slides and stained with haematoxylin and eosin (H&E) to evaluate general tissue organization. Masson’s Trichrome staining was performed to differentiate collagen, muscle and mineralized layers. Stained sections were analysed using a DM2000 microscope coupled to a DFC480 digital camera (Leica). Image J-1.51k (http://rsbweb.nih.gov/ij/ ) was used to estimate the number of mucous cells in the epithelia spanning 3 scales, every 4th section (15 μm) of skin in 4 sections/individual. In the gut, the abundance of mucous cells, the muscle layer thickness and the lamina propria thickness in three different positions (crypt, mid-point and tip) of 4 villi per section was determined in 3–4 sections/individual.
8
Daphnia magna Microscopy Techniques
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Samples for electron microscopy analysis of cross sections of D. magna were prepared as follows: the specimens were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for 1 hour at 4 °C, and post-fixed with a mixture of 1% osmium tetroxide. After three water washes, samples were dehydrated in a graded ethanol series and embedded in epoxy resin (Sigma-Aldrich). Semi-thin sections (1 µm) were obtained by an Ultrotome V (LKB) ultramicrotome, and counterstained with toluidine blue. D. magna specimens were observed by an XL 30 ESEM TMP microscope (Philips Electron Optics, Eindhoven, The Netherlands), equipped with an integrated energy dispersive X-ray spectrometer (XRF-EDS, EDAX system, Mahwah, NJ, USA). Moreover, D. magna cross sections (transversal and longitudinal) were analyzed by optical microscopy using a Leica DMR light microscope. Furthermore, living D. magna were filmed using a Leica MZ16 stereomicroscope equipped with a Leica DFC-480 digital camera.
9
Quantitative Immunohistochemical Analysis of 8-OHdG
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Formalin-fixed paraffin-embedded sections were deparaffinized and boiled for 20 min in 10mM citrate buffer (pH 6.0). Sections were washed in TBST and exposed to 0.3% H 2 O 2 for 5 min to quench endogenous peroxidases. Immunohistochemistry was performed using the following antibody: rabbit anti-8OHdG polyclonal antibody (1:100, BIOSS, Woburn, MA, USA). Concentration-matched rabbit IgG was used as an isotype-negative control. The sections were blocked with Dako proteinblock (Dako, Carpinteria, CA, USA) for 10 min and incubated with primary antibody overnight. The slides were then incubated with horseradish peroxidase anti-rabbit Envision-system followed by a 3.3'-diaminobenzidine (DAB) substratechromogen solution (Dako) and counterstained with Harris hematoxylin. The slides were examined using a Leica photomicroscope linked to a DFC 480 digital camera (Leica, Wetzlar, Germany).
The quantitation was performed by capturing 6-10 non-overlapping fields of renal cortex from stained sections. Areas of brown staining reflecting 8-OHdG were highlighted using a selective color tool and the proportional area of the field with their respective color range was quantified using Image J.
The quantitation was performed by capturing 6-10 non-overlapping fields of renal cortex from stained sections. Areas of brown staining reflecting 8-OHdG were highlighted using a selective color tool and the proportional area of the field with their respective color range was quantified using Image J.
10
Immunofluorescence Staining of Ciona Juveniles
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After rehydration, C. robusta juveniles were treated with pure acetone for 7 min and then with 1% Triton X-100 in PBS. Non-specific binding sites were blocked with the PBDT solution (PBS with 0.1% Tween 20, 1% BSA -Bovin Serum Albumin, 1% DMSO -Dimethyl sulfoxide, 0.2% Sheep serum) for 2 hours at room temperature before the over-night incubation at 4°C with the primary antibodies anti-Ci-VGlut (Horie, Kusakabe, & Tsuda, 2008) (link) or anti-GABA (Anti-ɤ-Aminobutyric acid) (both IgG diluted 1:100) (Table 2). Excess of primary antibody was washed off with PBT. Juveniles were then incubated with secondary antibody anti-mouse FITC (Calbiochem, cat. No. 401234, lot d21719 ) at 1:100 in PBDT. After three washes in PBT (10 min each), nuclei were stained with DAPI (1 µg/ml in H 2 O mQ) for 5 minutes. Samples were washed again with PBT, mounted in 70% glycerol in PBT, observed under Leica 5000 B microscope, and photographed with the Leica DFC 480 digital camera.
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