To measure vascular reactive oxygen species production, 5 μm sections of the thoracic aorta were exposed to dihydroethidium (DHE), following the previously described method [11 (link)]. Fresh, unfixed aorta samples were placed in optimal cutting temperature (OCT) compound (Sakura Finetek USA Inc., Torrance, CA) and frozen at − 80 °C. Ring segments were cut into 30 μm sections using a cryostat (Bright Instrument Company, Huntingdon, Cambridgeshire, England; model OTF) and placed on a glass slide. Sections were incubated in PBS for 30 min at 37 °C, and then DHE (2 μm) was topically applied. Coverslips were applied, and the sections were further incubated at 37 °C in a light-protected humidified chamber for 30 min. Sections were then rinsed in PBS, and fluorescence was detected using a 585-nm filter using an Olympus® inverted system microscope (Olympus America Inc.; model DP71). Images were photographed using an Olympus® digital camera (Olympus America Inc., model DP71) and analyzed in a blinded manner using ImageJ 1.42.
Model dp71
Manufactured by Olympus
Sourced in United States
The Olympus Model DP71 is a digital microscope camera designed for capturing high-quality images in microscopy applications. It features a 12.8-megapixel CCD sensor and can capture images at a resolution up to 4140 x 3096 pixels. The camera is capable of delivering real-time image previewing and fast data transfer through a FireWire interface.
Automatically generated - may contain errors
Lab products found in correlation
Dp controller, by Olympus (2 mentions)
Intravital microscope, by Olympus (2 mentions)
Model bx51wi, by Olympus (2 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Anti α sma, by R&D Systems (1 mentions)
Anti versican, by Abcam (1 mentions)
Xanthurenic acid, by Merck Group (1 mentions)
5 protocols using model dp71
1
Vascular Reactive Oxygen Sensing
2
Intravital Microscopy of Microvessels
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The animal preparation was transferred to the stage of an Olympus intravital microscope (Model BX51WI, Center Valley, PA) linked to a charge-coupled device (CCD) Olympus video camera (Model DP71). Microvessels were visualized using a 20× water-immersion objective. Video images were viewed on a high-resolution color monitor and digitally recorded via DP Controller (Olympus, Center Valley, PA) for off-line analysis (Image J) [24 (link),41 (link)].
3
Intravital Microscopy of Microvessels
4
Immunohistochemical Profiling of Hair Follicle Cells
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Immunohistochemical staining was performed according
to standard protocols. In brief, dermal papilla and hair
keratinocytes were fixed in 4% paraformadehyde solution
and permeabilized using Triton X-100 (MERK, Germany,
0.2%). After washing with PBS/Tween (PBST) 0.05%
(Sigma, USA), the cells were incubated in primary
antibodies, including anti-versican (Abcam, USA), anti-α-SMA (R&D, USA) and anti-K15 (Abcam, USA),
overnight at 4ºC. After washing, dermal papilla and hair
keratinocytes were incubated in secondary antibodies for
one hour at 37ºC. Specific cell markers were ultimately
checked with fluorescence microscope Olympus Model
DP71. DAPI was also applied for nuclear staining.
to standard protocols. In brief, dermal papilla and hair
keratinocytes were fixed in 4% paraformadehyde solution
and permeabilized using Triton X-100 (MERK, Germany,
0.2%). After washing with PBS/Tween (PBST) 0.05%
(Sigma, USA), the cells were incubated in primary
antibodies, including anti-versican (Abcam, USA), anti-α-SMA (R&D, USA) and anti-K15 (Abcam, USA),
overnight at 4ºC. After washing, dermal papilla and hair
keratinocytes were incubated in secondary antibodies for
one hour at 37ºC. Specific cell markers were ultimately
checked with fluorescence microscope Olympus Model
DP71. DAPI was also applied for nuclear staining.
5
Quantifying Male Gamete Activation in Malaria
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The male gamete activation assay was performed following a slight modification of an established protocol (Delves et al., 2013 (link)(Delves et al., , 2016)) (link). In brief, in vitro gametocyte culture was set up as described above. On day 13 of the culture, the gametocytes were treated with P218 or other antimalarial compounds at 37 °C for 48 hours.
Half of the media supplemented with antimalarial compounds was changed and then incubated for another 24 hours before exflagellation readout. The male gametocyte exflagellation was induced using ookinetes culture medium (RPMI 1640 with 25 mM HEPES, 2 mM glutamine (Sigma, Cat no. G7513), supplemented with 100 µM xanthurenic acid (Sigma, Cat. no. D120804) and observed with hemocytometer under a light-contrast microscope. For each replicate, eight fields of view were recorded with Olympus video camera system (Model DP71) then the total number of exflagellation sites/1,000 red blood cells was calculated then exflagellation inhibition was compared to the DMSO control.
Half of the media supplemented with antimalarial compounds was changed and then incubated for another 24 hours before exflagellation readout. The male gametocyte exflagellation was induced using ookinetes culture medium (RPMI 1640 with 25 mM HEPES, 2 mM glutamine (Sigma, Cat no. G7513), supplemented with 100 µM xanthurenic acid (Sigma, Cat. no. D120804) and observed with hemocytometer under a light-contrast microscope. For each replicate, eight fields of view were recorded with Olympus video camera system (Model DP71) then the total number of exflagellation sites/1,000 red blood cells was calculated then exflagellation inhibition was compared to the DMSO control.
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