Endogenous amyloids from fractionated or unfractionated SP were visualized by staining samples with Proteostat and/or pFTAA, using approaches previously described (Usmani et al., 2014 (link)). Briefly, fractionated or unfractionated SP were incubated with Proteostat and/or pFTAA for 15 min at room temperature, and then transferred into an Ibidi Chamber Slide (#80826 from GmbH). Stained samples were imaged on a Zeiss LSM710 AxioObserver confocal microscope equipped with a Plan-Apochromat 63/1.40 oil objective lens and Zen-Software v2010 (Zeiss, Germany). Spermatozoa were simultaneously imaged by DIC using transmitted light detector (T-PMT) and appropriate condenser settings. Where indicated, spermatozoa were additionally visualized by staining with Hoechst 33342.
Lsm 710 axioobserver confocal microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 710 AxioObserver confocal microscope is a high-performance imaging solution designed for advanced biological and materials research. It features a modular design that allows for customization to meet specific experimental requirements. The microscope utilizes laser scanning technology to capture high-resolution, optical sectioning images with excellent signal-to-noise ratio and contrast.
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Lab products found in correlation
Prolong gold dapi, by Thermo Fisher Scientific (2 mentions)
Anti lamp1, by Merck Group (2 mentions)
Anti rab7 d95f2, by Cell Signaling Technology (2 mentions)
Mabt837, by Merck Group (2 mentions)
Fluoromount g, by Southern Biotech (2 mentions)
Zen software v2010, by Zeiss (1 mentions)
Stellaris eight confocal microscope, by Leica (1 mentions)
Cm1850, by Leica (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Zen 2011, by Zeiss (1 mentions)
Fm4 64 dye, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 40x 1.4 oil dic m27, by Zeiss (1 mentions)
Pentr d topo vector, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Ix81 microscope, by Zeiss (1 mentions)
Matrigel, by Beckman Coulter (1 mentions)
Rat tail collagen 1, by Thermo Fisher Scientific (1 mentions)
Cell recovery solution, by Corning (1 mentions)
Matrigel, by Corning (1 mentions)
18 protocols using lsm 710 axioobserver confocal microscope
1
Visualizing Endogenous Amyloids in Spermatozoa
2
Spatiotemporal Analysis of Cry2 Activation
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Time-lapse microscopy of activated Cry2 fusions was performed on a Zeiss LSM 710 AxioObserver confocal microscope with full incubation chamber in conjunction with the Zeiss ZEN software. Cry2 translocation experiments were carried out at 25°C, while experiments examining cell motility and polarity were carried out at 37°C and in 5% CO2. EGFP, Venus, and mCherry were visualized with 488, 514, and 561 nm laser excitation, respectively, through either a 40x or 63x oil immersion objective. Whole field Cry2 activation was achieved using 450 or 488 nm illumination. Spatially defined activation was carried out under whole cell imaging of Venus with a 514 nm laser in conjuction with focal illumination using the FRAP application with 458 or 488 nm laser light at 1–5% power and 10 μs dwell time per pixel in a 10 μm diameter region. For whole field observation of lamellipodial induction and polarity establishment, cells were excited every 13 seconds with 488 nm light and mCherry was imaged simultaneously.
3
Cryosectioning and Imaging of Insect Heads
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Control and cut knockdown fly and beetle heads were fixed in 4% formaldehyde overnight at 4°C. These tissues were washed three times with PBS and then cryoprotected overnight at 4°C in sucrose solutions of increasing concentrations (20%, 40%, and 60%). The samples were mounted in Neg50, flash frozen in liquid nitrogen, and cryosectioned at ∼18 µm (Leica CM 1850). The sections were dried, rinsed in PBS, and stained with Phalloidin (following manufacturer’s instruction) and mounted with Fluoromount containing DAPI (Thermo Fisher). Z-stacks for D. melanogaster samples were obtained at a resolution of 1,024 × 1,024 pixels using a Leica Stellaris eight confocal microscope with a ×40 objective. T. marmoratus samples were imaged using a Zeiss LSM 710, AxioObserver confocal microscope with a ×40 objective. The entire eye was tile scanned with a constant pixel size of 0.69 µm. All images were processed using ImageJ, and brightness and contrast were adjusted using Adobe Photoshop 2022.
4
Immunofluorescence Staining of Cultured Cells
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Cells were plated on glass coverslips and grown for 48 h before any starvation or drug treatment. After treatment, cells were fixed with 4% (wt/vol) paraformaldehyde (Electron Microscopy Sciences) for 15 min. Cells were permeabilized using 0.1% Triton X-100 in PBS for all endogenous protein staining except when LC3 staining was required. For endogenous LC3 staining, cells were first fixed in 4% paraformaldehyde followed by 100% methanol for 10 min. Cells were blocked in 1% BSA in PBS for 20 min. Primary antibody staining was performed for 1 h in 1% BSA in PBS, followed by two PBS washes for 5 min each. Secondary antibody staining was performed for 30 min using Alexa Fluor Dyes (Molecular Probes) in 1% BSA in PBS. Coverslips were mounted onto glass slides using ProLong Gold antifade reagent with or without DAPI for nuclear staining. Images were taken using Zeiss LSM 710 Axio Observer confocal microscope with 63× objective lens. Image postprocessing was performed using Adobe Photoshop for inset enlargement and image alignment. Live-cell montage was first generated in ImageJ before further separation of RGB channels in Adobe Photoshop.
5
Immunofluorescence Characterization of Pituitary Adenoma
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Six-micrometer sections of paraffin-embedded nude mice tumors, previously fixed with Bouin-Hollande solution, deparaffinized and rehydrated, and primary human pituitary adenoma cultures were incubated (4 C, 18 h) with antibodies against p16INK4a (1:50, Santa Cruz Technologies), pRb (1:50, Cell Signalling Technology) or c-myc (1:50, Santa Cruz Technologies). After washing with PBS, samples were incubated (45 min, room temperature) with secondary antibody Alexa Flour 647. Nuclei were stained by DAPI. MtT/S clones were fixed in 4% PFA and nuclei were stained with DAPI. All samples were mounted with Mowiol mounting medium and observed with a LSM 710 AxioObserver confocal microscope (Carl-Zeiss). For tumor sections, six independent pictures for each condition were taken. Images were acquired with ZEN 2011 software (Carl-Zeiss) and analyzed in ImageJ.
6
Spatiotemporal Analysis of Cry2 Activation
7
FRAP Analysis of Halo-CTCF Dynamics
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FRAP was performed exactly as previously described19 (link) for C59 mESCs (Halo-CTCF; Rad21-SNAPf) and C59D2 mESCs (ΔRBRi-Halo-CTCF; Rad21-SNAPf) on an inverted Zeiss LSM 710 AxioObserver confocal microscope (330 frames, 2 sec between frames, 100 nm pixels, circular bleach spot with 10 pixel radius). We performed 3 biological replicates recording a total of 18 cells for C59 Halo-CTCF and 18 cells for C59D2 ΔRBRi-Halo-CTCF and analyzed the data as previously described19 (link). As demonstrated previously19 (link),54 , Halo-CTCF and ΔRBRi-Halo-CTCF fall in the “reaction dominant” regime, where the recovery depends only on the kOFF and we therefore fit the FRAP recoveries to the reaction dominant model below:
We interpret the slower rate as specific binding to cognate sites. The fits are shown inSupplementary Fig. 15 .
We interpret the slower rate as specific binding to cognate sites. The fits are shown in
8
FISH Protocols for Arabidopsis and Zebrafish
9
Transient Expression of MWL-1 and MWL-2 in Populus
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Genes encoding MWL-1 and MWL-2 were amplified from Arabidopsis Col-0 cDNA using gene-specific forward primers and modified reverse primers where the stop codon was removed (S1 Table ). The PCR products were cloned into the pENTR™/D-TOPO® vector (Invitrogen™), sequenced verified, and thereafter recombined into the transient expression vector pSAT-DEST-GFP-N1B (CD3-1654, TAIR) downstream a double 35S CaMV promoter and in-frame with a C-terminal Green Florescent Protein (GFP). Protoplast isolation and PEG-calcium transfection was carried out in Populus alba × tremula, P717 protoplasts using the method described by Guo et al. (2012) [13 (link)]. FM® 4–64 dye (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide; ThermoFisher Scientific) was used as a positive membrane marker. Florescence was detected 24-hours post-transfection using a Zeiss LSM 710 AxioObserver confocal microscope with the Plan-Apochromat 40x/1.4 Oil DIC M27. Excitation wavelength for GFP and FM® 4–64 was 488 and 515 nm, respectively, while emission was detected at 495–540 and 640 nm, respectively. Images were processed with Zen 2 lite blue edition (Zeiss).
10
Drosophila Pupal Eye Dissection and Imaging
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Pupal eyes (at day 2 APF) were dissected and processed as described for D. melanogaster eyes, except that the tissue was counterstained with phalloidin (Alexa Fluor 647, Thermo Fisher) instead of anti-N-cadherin (due to a lack of cross-reactivity). The anti-Cut antibody previously has been successfully used in beetles (Burns et al., 2012 (link); Miguel et al., 2016 (link)). Z-stacks were acquired using a Zeiss LSM 710, AxioObserver confocal microscope with a ×40 objective at a resolution of 512 × 512 pixels and a pixel size of 0.11 µm. All images were processed using ImageJ, and the brightness and contrast were adjusted using Adobe Photoshop 2022.
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