At 24, 48, 72, 96, 120, and 168 h pibm, midguts were dissected and fixed in 4% para-formaldehyde dissolved in 1x PBS. Fixed midguts were permeabilized using PBS-T (1x PBS containing 0.2% Triton X-100 and 1% BSA), before a 1 h incubation at room temperature (RT) with the primary monoclonal antibody Anti-Flavivirus Group Antigen D1-4G2-4-15 (ATCC #: VR-1852), diluted 1:500 in PBS-T. Following several washes with PBS-T, the secondary monoclonal antibody (goat anti-mouse IgG labeled with Alexa Fluor 594, Abcam # ab150120) was applied for 1 h at RT at a 1:500 dilution in PBS-T together with Alexa Fluor Phalloidin 488 (Invitrogen, Carlsbad, CA, USA) diluted 1:1000. Cell nuclei were stained with 1 µg/mL DAPI (Invitrogen) for 10 min. Midguts were then washed three times with PBS before being mounted on six-well printed slides, using Fluoromount G mounting medium (Electron Microcopy Sciences, Hatfield, PA, USA). Samples were viewed under an inverted spectral confocal microscope (TCP SP8 MP, Leica Microsystems, Wetzlar, Germany) at the Molecular Cytology Core of the University of Missouri.
Tcp sp8 mp
Manufactured by Leica
Sourced in Germany
The TCP SP8 MP is a high-performance confocal microscope system designed for multi-photon imaging. It features a tunable pulsed laser source and advanced optical components to enable deep tissue imaging and enhanced resolution. The system is capable of capturing detailed images of biological samples at the cellular and sub-cellular level.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Fluoromount g mounting medium, by Electron Microscopy Sciences (2 mentions)
Lacv monoclonal antibody 8c2.2, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Abcam (1 mentions)
4 protocols using tcp sp8 mp
1
Flavivirus Midgut Immunostaining Protocol
2
Mosquito Midgut Nanoparticle Uptake
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Seven days-old HWE mosquitoes were deprived of food for one day and water for 4 h before receiving a proteinmeal consisting of 20% BSA using artificial glass feeders as described above. Fully engorged mosquitoes were selected and their midguts dissected at 24, 48, 60, 72, 96, 120, and 168 h post-feeding (pf). Dissected midguts were soaked for 2 h in a suspension consisting of 5 nm FITC labeled gold-nanoparticles (1.99 × 109 nanoparticles/mL) and 50 nm Cy3 labeled gold-nanoparticles (1.99 × 109 nanoparticles/mL) (Nanopartz, Loveland, CO, USA). Midguts were then washed three times with 1x PBS before being stained with 1 µg/mL DAPI for 10 min. Following several washes with PBS, midguts were mounted on six-well printed slides and viewed under an inverted spectral confocal microscope (TCP SP8 MP, Leica Microsystems). A 3D-image was obtained from a midgut sample of a proteinmeal-fed female at 72 h pf. 3D-image reconstruction along the x, y, and z axes was performed using the LAS X 3D imaging software (Leica, Wetzlar, Germany).
3
Visualizing LACV Infection in Mosquito Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Midguts and ovaries were dissected at various time points following challenge with an LACV-containing bloodmeal and fixed in 4% paraformaldehyde–phosphate buffer solution (PBS) (Gibco, ThermoFisher Scientific) at 4 °C for a minimum of 30 min. Tissues were permeabilized in PBS-T (PBS, 1% BSA, 0.2% Triton X-100) at room temperature (RT) for 1 h. Tissues were then incubated overnight at 4 °C in PBS-T containing LACV monoclonal antibody (8C2.2) (Invitrogen, ThermoFisher Scientific (Waltham, MA, USA)) at a 1:200 dilution. Samples were then washed three times for twenty minutes each with PBS-T, then incubated with fluorescent-labeled secondary antibody (Alexa Fluor 594, Abcam: ab150120, Cambridge, UK) at a dilution of 1:500 and counter-stained with Phalloidin (AlexaFluor 488, Invitrogen, ThermoFisher Scientific) at 1:100 dilution for 1 h at RT. Cell nuclei were stained with DAPI (Invitrogen) at 1 μg/mL for ten minutes at RT. Tissues were washed three times again with PBS and mounted on six-well slides using Fluoromount G mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA). Tissues were imaged using an inverted spectral confocal microscope (TCP SP8 MP, Leica Microsystems, Wetzlar, Germany) at the Molecular Cytology Core of the University of Missouri.
4
Immunohistochemical Analysis of Zika Virus in Mosquito Midguts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Midguts were dissected at 5, 7, 8, 9, and 11 days following challenge with a ZIKV-containing blood-meal and fixed in 4% paraformaldehyde diluted in phosphate buffer solution (PBS; Gibco, ThermoFisher, Waltham, MA, USA) at 4°C for at least 30 minutes. Midguts were then permeabilized in PBS-T (PBS with 1% BSA and 0.2% Triton-X-100) for 1 hour at room temperature (RT) followed by incubation at 4°C overnight with the primary (monoclonal) antibody Anti-Flavivirus Group Antigen D1-4G2-4-15 (ATCC: VR-1852) diluted 1:500 in PBS-T. Samples were then washed three times with PBS-T with each wash step lasting 5 minutes. The secondary (monoclonal) antibody, goat anti-mouse IgG labelled with Alexa Fluor 594 (Abcam: ab150120) was added at a 1:500 dilution in PBS-T for 1 hour at RT in the dark, along with Alexa Fluor Phalloidin 488 (Invitrogen, Carlsbad, CA, USA) at a 1:1000 dilution. Cell nuclei were stained with DAPI (Invitrogen) at 1μg/mL for 10 minutes at RT. Midguts were washed three times with PBS-T again and mounted onto six-well slides using Fluoromount G mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA). Samples were imaged using an inverted spectral confocal microscope (TCP SP8 MP, Leica Microsystems, Wetzlar, Germany) at the Molecular Cytology Core of the University of Missouri.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!