Antisense RNA probes labeled with either digoxigenin-11 rUTP or fluorescein-12 rUTP (Roche) were synthesized as described by Sive et al. (2000) for the following genes: tubb2b (linearized with BamHI, transcribed with T7) (Klein et al., 2002 (link)), sox2 (linearized with EcoRV, transcribed with T7) (Huyck et al., 2015 (link)), and pcna (linearized with XhoI, transcribed with T7) (Huyck et al., 2015 (link)). PCNA in situ hybridization (ISH) was selected based on reports in the literature suggesting that it represents an effective and accurate tool for assessing dynamic changes in proliferation (Muskhelishvili et al., 2003 (link); Kohler et al., 2005 (link)). Whole-mount chromogenic in situ hybridization (ISH) using nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/ BCIP) alkaline phosphatase substrates was performed as described by Sive et al. with minor modifications (Sive et al., 2000 ). Embryos were photographed using an Olympus SZH10 microscope and an Olympus DP71 camera or Nikon SMZ800N microscope and Nikon DSi-R2 camera. Global image adjustments were made using Adobe Photoshop CC to correct brightness, contrast, and color balance.
Dp71 camera
Manufactured by Nikon
The DP71 camera is a high-performance digital microscope camera designed for scientific and industrial applications. It features a 12.8-megapixel CCD sensor and is capable of capturing images with a resolution of up to 4140 x 3096 pixels. The camera is compatible with a variety of microscope systems and can be used for a range of imaging tasks, including brightfield, darkfield, and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Antisense rna probes, by Roche (2 mentions)
Digoxigenin 11 rutp, by Roche (2 mentions)
Fluorescein 12 rutp, by Roche (2 mentions)
Smz800n microscope, by Nikon (2 mentions)
Dsi r2 camera, by Nikon (2 mentions)
Szh10 microscope, by Olympus (2 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Igg free albumin, by Merck Group (1 mentions)
Rabbit anti gfap, by Thermo Fisher Scientific (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Vectashield medium, by Vector Laboratories (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
4 protocols using dp71 camera
1
In Situ Hybridization for Proliferation Markers
2
In Situ Hybridization for Proliferation Markers
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Antisense RNA probes labeled with either digoxigenin-11 rUTP or fluorescein-12 rUTP (Roche) were synthesized as described by Sive et al. (2000) for the following genes: tubb2b (linearized with BamHI, transcribed with T7) (Klein et al., 2002 (link)), sox2 (linearized with EcoRV, transcribed with T7) (Huyck et al., 2015 (link)), and pcna (linearized with XhoI, transcribed with T7) (Huyck et al., 2015 (link)). PCNA in situ hybridization (ISH) was selected based on reports in the literature suggesting that it represents an effective and accurate tool for assessing dynamic changes in proliferation (Muskhelishvili et al., 2003 (link); Kohler et al., 2005 (link)). Whole-mount chromogenic in situ hybridization (ISH) using nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/ BCIP) alkaline phosphatase substrates was performed as described by Sive et al. with minor modifications (Sive et al., 2000 ). Embryos were photographed using an Olympus SZH10 microscope and an Olympus DP71 camera or Nikon SMZ800N microscope and Nikon DSi-R2 camera. Global image adjustments were made using Adobe Photoshop CC to correct brightness, contrast, and color balance.
3
Immunofluorescence Staining of Mouse Brain Sections
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The half of each brain obtained as described above was fixed in 4 % buffered formalin at 4 °C overnight. Free-floating 30 μm-thick mouse brain sections were processed as previously described [33 (link)]. After antigen retrieval by incubating in citrate buffer (0.01 M citric acid, 0.05 % Tween 20, pH 6.0) at 70 °C for 50 min, samples were thoroughly washed several times with TBS and blocked with a solution of 2 % IgG-free albumin (Sigma) in TBS for 20 min at room temperature. Brain sections were then incubated overnight at 4 °C with rabbit anti-GFAP (Invitrogen) or anti-Iba-1 (WAKO, VA) polyclonal antibody in TBS-2 % BSA to detect astrocytes or microglia, respectively. After washing, sections were incubated for 1 h at room temperature with AlexaFluor 594 goat anti-rabbit IgG (Molecular Probes, OR) diluted in TBS-2 % BSA. Samples were mounted onto glass slides in Vectashield medium (Vector Laboratories, CA) containing DAPI for nuclei imaging. Samples were viewed on an Olympus Ix51 microscope equipped with a DP71 camera (Nikon Instruments Inc., NY).
4
Quantifying Cardiac Fibrosis by Histology
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Hearts were fixed overnight in 4% PFA and embedded in paraffin. To determine the fibrotic area, the whole heart was transversely sectioned (7 µm), and sections were selected for analysis every 70 to 140 µm. Masson’s trichrome staining was performed according to standard procedures. Images were acquired with a BX51 microscope (Olympus), a DP71 camera (Nikon), and cellSens software. The fibrotic area was measured in the section with the largest amount of fibrotic tissue as a percentage of the total section. Likewise, the percentage of the fibrotic region occupied by CMs was quantified in the same section. The calculations were done with Metamorph (Molecular Devices).
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