To determine the cytotoxic doses, and to determine the highest tolerated dose of our mixture we performed a titration of the GWI toxicant mixture with a LIVE/DEAD® (Thermo Fisher Scientific) cell imaging kit. Viability is detected by intracellular esterase activity that converts calcein AM to fluorescent calcein and cell death is indicated by entry of fluorescent BOBO-3 through degrading cell membranes. The liquid and the lyophilized components are combined and added in a 1:1 mixture to the cells and incubated for 15 min at room temperature. The live-cell component fluoresces with (ex/em 488 nm/515 nm) and the dead cell component fluoresces with (ex/em 570 nm/602 nm). Cells were imaged with an Olympus Fluoview 3000 laser scanning confocal microscope and counted with ImageJ.[37 (link)] Total live and total dead cells were counted from 4 fields from each well. Data are reported as percent dead.
Fluoview 3000 laser scanning confocal microscope
Manufactured by Olympus
The Fluoview 3000 Laser Scanning Confocal Microscope is a high-performance imaging system designed for advanced fluorescence microscopy. It features a multi-laser configuration, enabling the simultaneous excitation of multiple fluorophores. The microscope's confocal design allows for the capture of high-resolution, optical sectioning images with improved contrast and reduced background interference.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti embryonic lethal abnormal vision, by Developmental Studies Hybridoma Bank (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 568, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
Mouse anti dlg, by Developmental Studies Hybridoma Bank (1 mentions)
Goat anti rat igg, by Jackson ImmunoResearch (1 mentions)
Donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Mouse anti 6e10, by Fortrea (1 mentions)
4 protocols using fluoview 3000 laser scanning confocal microscope
1
Cytotoxicity Assessment of GWI Mixture
2
Validating Canoe Knockdown in Drosophila
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate Canoe knock down by canoe RNAi, stage 14 embryos expressing daughterless-Gal4 and either UAS-mcherry-RNAi or UAS-canoe-RNAi were dechorionated as above and fixed for 30 min in a 1:1 mix of heptane and 4% formaldehyde in PBS. Embryos were devitellinized manually. Staining was with primary antibodies rabbit anti-Canoe (1:1000, gift from Mark Peifer) and mouse anti-Dlg (1:500, Developmental Studies Hybridoma Bank) and secondary antibodies goat anti-rabbit Alexa 488 (1:500, Invitrogen) and goat anti-mouse Alexa 568 (1:500, Invitrogen).
Embryos were mounted in Prolong Gold (Molecular Probes) between two coverslips.
Stained embryos were imaged at 60X magnification on an Olympus FluoView 3000 laser scanning confocal microscope. Canoe intensities were normalized to the Dlg signal for quantification of Canoe knockdown.
Embryos were mounted in Prolong Gold (Molecular Probes) between two coverslips.
Stained embryos were imaged at 60X magnification on an Olympus FluoView 3000 laser scanning confocal microscope. Canoe intensities were normalized to the Dlg signal for quantification of Canoe knockdown.
3
Immunostaining of Drosophila Imaginal Discs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Eye-antennal imaginal discs were dissected from the third instar larvae in cold 1× phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in 1× PBS for 20 min, and then quickly washed once in 1× PBS. It was followed by three washes in 1× PBST. The tissues were stained with a combination of antibodies following a previously published protocol [42 (link), 52 (link)]. The primary antibodies used were rat anti-Embryonic Lethal Abnormal Vision (ELAV) (1:100; Developmental Studies Hybridoma Bank, DSHB), mouse anti-Discs-large (Dlg) (1:100; DSHB), and mouse anti-Chaoptin (24B10) (1:100; DSHB) [53 (link)]. Secondary antibodies (Jackson Laboratory) used were goat anti-rat IgG conjugated with Cy5 (1:250), and donkey anti-mouse IgG conjugated with Cy3 (1:250). The tissues were mounted in the antifading agent: Vectashield (Vector Laboratories). The immunofluorescent images were captured at 20× magnification by using Olympus Fluoview 3000 Laser Scanning Confocal Microscope [54 ]. All final figures were prepared using Adobe Photoshop software.
4
Immunofluorescence Labeling of Drosophila Eye-Antennal Discs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Eye-antennal imaginal discs were dissected from the third instar larvae in cold 1X phosphate buffered saline (PBS), fixed in 4% paraformaldehyde in 1X PBS for 20 min, and stained using a standardized protocol [57 (link)]. Primary antibodies used were rat anti-Embryonic Lethal Abnormal Vision (ELAV) (1:100; Developmental Studies Hybridoma Bank, DSHB, Catalogue number #7E8A10), mouse anti-Discs-large (Dlg) (1:100; DSHB, Catalogue number #4F3), mouse anti-acetylated Tubulin (1:100; DSHB, Catalogue number #12G10), mouse anti-Chaoptin (24B10) (1:100; DSHB, Catalogue number #24B10) [58 (link)], mouse anti-6E10 (1:100; Covance, Catalogue number #SIG-39320) and rabbit Mnat9 (1:100; a gift from Dr. Kwang-Wook Choi) [49 (link)]. Secondary antibodies (Jackson Laboratory) used were goat anti-rat IgG conjugated with Cy5 (1:250; Catalogue number #112-175-143), donkey anti-mouse IgG conjugated with Cy3 (1:250; Catalogue number #715-165-150) and donkey anti-rabbit IgG conjugated with Cy3 (1:250; Catalogue number #711-165-152). We mounted the tissues in antifading agent Vectashield (Vector Laboratories). The immunofluorescent images were captured at 20X magnification using Olympus Fluoview 3000 Laser Scanning Confocal Microscope [59 ]. All final figures were prepared using Adobe Photoshop software.
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!