For the observation of propidium iodide (PI) stained roots, 5-day-old seedlings grown on ½MS plates were mounted in ddH2O containing 10 μM PI for 20 min before imaged with the Leica SP8 confocal microscope. Images were captured at 543 nm laser excitation and 578–700 nm emission. For the observation of TAMRA-SCOOP12 labeled roots and protoplasts, 5-day-old seedlings grown on ½MS plates or protoplasts isolated from leaves of 4-week-old soil-grown plants were treated with 100 nM TAMRA-SCOOP12 with or without 1 μM SCOOP12 or flg22 for 5 min in liquid ½MS or W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.7), followed by washing with ½MS or W5 solution for three times before imaged with the Leica SP8 confocal microscope. Images were captured at 552 nm laser excitation and 570–620 nm emission. To observe SCOOP12-GFP in Arabidopsis pSCOOP12::SCOOP12-GFP/scoop12 transgenic plants, detached leaves were imaged under the Leica SP8 confocal microscope directly or 3 min after 5% sodium chloride (NaCl) treatment. Images were captured at 472 nm laser excitation and 493–547 nm emission. For all the observations, the pinhole was set at one Airy unit, and the imaging processing was carried out by using the Leica Application Suite X (LAS X) software.
Sp8 confocal microscope
Manufactured by Leica
Sourced in Germany, United States, Japan, Italy, France, United Kingdom, China, Switzerland, Canada, Portugal
The Leica SP8 confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a state-of-the-art confocal architecture that enables high-resolution, real-time imaging of fluorescently labeled samples. The SP8 offers precise control over laser excitation, detector settings, and optical parameters to optimize image quality and data acquisition.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (101 mentions)
Las x software, by Leica (61 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (40 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (38 mentions)
Vectashield, by Vector Laboratories (36 mentions)
Hoechst 33342, by Thermo Fisher Scientific (30 mentions)
Triton x 100, by Merck Group (28 mentions)
Lsm 710 confocal microscope, by Zeiss (21 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (20 mentions)
Las af software, by Leica (18 mentions)
Las x, by Leica (18 mentions)
Bovine serum albumin (bsa), by Merck Group (18 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (17 mentions)
Application suite x, by Leica (17 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (15 mentions)
Fluoromount g, by Southern Biotech (15 mentions)
Cryostat, by Leica (15 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (15 mentions)
Application suite x software, by Leica (14 mentions)
Vectashield mounting medium, by Vector Laboratories (13 mentions)
2 556 protocols using sp8 confocal microscope
1
Fluorescent Labeling and Imaging of Plant Roots
2
Imaging and ROS Detection Assay
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The expression of gstD-GFP and Drs-GFP as well as the distribution of TE-SPc nanoparticles were monitored under a Leica SP8 confocal microscope. The cellular ROS level was examined by DHE staining [93 (link)]. Fluorescent photos were captured with Leica SP8 confocal microscope. The laser intensity and exposure time was set at the same value when samples from the control group and SPc treatment group were photographed.
3
Eupyrene Sperm Analysis in Spermatophores
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Eupyrene sperm in the spermatophores at 5 h after mating were collected and then centrifuged at 1000 × g for 10 min. The eupyrene sperm pellet was resuspended and a 20 μL drop of samples was placed on a microscope slide to air-dry. Samples were fixed with acetone (4°C) for 15 min and then stained with FITC-PSA (100 μg mL -1 ; 37°C) for 60 min. Stained samples were observed using a Leica SP8 confocal microscope and at least 200 eupyrene sperm were evaluated per smear.
2.9 Comet assay DNA damage of eupyrene sperm in the spermatophores at 5 h after mating was detected by alkaline comet assay using the DNA Damage Assay Kit (Nanjing Jiancheng Bioengineering Institute). In brief, the eupyrene sperm suspension was combined with 0.7% low melting agarose and then immersed in lysis buffer (4°C) for 90 min. After washing in PBS buffer, the slides were immersed in alkaline electrophoresis buffer for 30 min and then electrophoresed for 25 min (25 V and 300 mA). Next, slides were washed with neutralization buffer and then treated with PI solution for 10 min. Stained slides were observed using a Leica SP8 confocal microscope (> 50 sperm per slide) and comet tail DNA (%) was quantified using Comet Assay Software Project 1.2.3 beta 1 (http://www. casp.of.pl/).
2.9 Comet assay DNA damage of eupyrene sperm in the spermatophores at 5 h after mating was detected by alkaline comet assay using the DNA Damage Assay Kit (Nanjing Jiancheng Bioengineering Institute). In brief, the eupyrene sperm suspension was combined with 0.7% low melting agarose and then immersed in lysis buffer (4°C) for 90 min. After washing in PBS buffer, the slides were immersed in alkaline electrophoresis buffer for 30 min and then electrophoresed for 25 min (25 V and 300 mA). Next, slides were washed with neutralization buffer and then treated with PI solution for 10 min. Stained slides were observed using a Leica SP8 confocal microscope (> 50 sperm per slide) and comet tail DNA (%) was quantified using Comet Assay Software Project 1.2.3 beta 1 (http://www. casp.of.pl/).
4
Quantifying Mitotic Cells in Intestine
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Dividing cells were marked with Phospho-Histone H3 staining (pH3) antibody and mitotic cells were counted in the anterior and posterior intestine using a Leica SP8 confocal microscope with a 40× objective. Representative images were taken using a Leica SP8 confocal microscope. Images were analyzed, processed, and compiled using Prism 8, Fiji [24 (link)], and Inkscape.
5
Cellular Localization of miR-21 in DRG
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Cellular localization of miR-21 in DRG was performed using the mature mouse miR-21 (mmu-miR-21a-5p) detection probe for ISH labeled by 5′- and 3′-FAM (Exiqon). In brief, animals were fixed via the ascending aorta with 0.1 M PBS containing 4% paraformaldehyde and 0.1% DEPC. DRGs were collected and then cut in a cryostat. DRG sections (15 µm) were treated with proteinase K (BosterBio) for 2 min at room temperature, and then washed in RNase-free PBS. After digestion with proteinase, sections were post-fixed with the 1% paraformaldehyde/PBS and then washed in DEPC-treated ultrapure water. Prehybridization procedures were performed under RNase-free conditions for 2 h at 40°C. Hybridization was performed with a hybridization probe specific to miR-21 at 42°C overnight in hybridization buffer (Jiang et al., 2016 (link)). Sections were then washed with 2× saline sodium citrate (SSC), 0.5× SSC, and 0.2× SSC buffers. The signal was detected with the Leica SP8 confocal microscope.
To identify the colocalization of miR-21 and TLR8, the above sections under miR-21 ISH were incubated overnight at 4°C with primary antibodies against TLR8. On the following day, a Cy3-conjugated secondary antibody was added and incubated for 2 h. The signal was detected with a Leica SP8 confocal microscope.
To identify the colocalization of miR-21 and TLR8, the above sections under miR-21 ISH were incubated overnight at 4°C with primary antibodies against TLR8. On the following day, a Cy3-conjugated secondary antibody was added and incubated for 2 h. The signal was detected with a Leica SP8 confocal microscope.
6
Visualizing Neurodegeneration in C. elegans
7
Histological and Fluorescence Analysis of Bone Remodeling
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Bone specimens were decalcified with 10% ethylenediaminetetraacetic acid for 14 days. The bone tissue was then embedded in paraffin and cut into 5-μm-thick sections. Haematoxylin and eosin (H&E) staining, safranin-fixed green staining, and Masson’s trichrome staining were performed. A Leica DM4000 microscope (Leica) was used to capture images. Immunofluorescence staining was performed on frozen sections, and images were acquired using an SP8 confocal microscope (Leica). Anti-CD31, collagen-1 (COL-1), and matrix metalloproteinase 13 (MMP13) antibodies were obtained from Abcam (China). Fluorescence in situ hybridization (FISH) was performed on frozen sections, and images were acquired using an SP8 confocal microscope (Leica). The probe of miR-19a-3p was obtained from Ruibo.
To observe dynamic bone formation, tetracycline (25 mg/kg; Aladdin, China), alizarin red (30 mg/kg, Aladdin), and calcein AM (10 mg/kg, Aladdin) were injected intramuscularly at one, two, and three weeks post-surgery. After the animal kill and specimen preparation, bone formation was observed using a confocal laser-scanning microscope (Leica).
To observe dynamic bone formation, tetracycline (25 mg/kg; Aladdin, China), alizarin red (30 mg/kg, Aladdin), and calcein AM (10 mg/kg, Aladdin) were injected intramuscularly at one, two, and three weeks post-surgery. After the animal kill and specimen preparation, bone formation was observed using a confocal laser-scanning microscope (Leica).
8
Imaging and Analysis of Protein Aggregation in C. elegans
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To obtain confocal z-stacks of transgenic C. elegans, animals were fixed with 4% paraformaldehyde and actin filaments were stained with phalloidin (Molecular Probes/Life Technologies, Grand Island, NY) as previously described [7 (link)]. Imaging was with a Leica SP8 confocal microscope (Wetzlar, Germany) using a 40X oil immersion objective. For Fluorescence Recovery after Photobleaching (FRAP) of Htt513(Q128) foci in living animals, day 1 adults were immobilized with 2mM levamisole, mounted on 2% agarose pads, and covered with a coverslip. FRAP was performed on a Leica SP8 confocal microscope with a 63X oil immersion objective (Wetzlar, Germany). Data were analyzed as previously described [31 (link)]. For analysis of aggregation over time, images were obtained with a Ziess Axio Observer A1 (Oberkochen, Germany) inverted compound fluorescence microscope using a 20X objective. Micrographs of small regions of the animal were stitched together manually to obtain images of whole animals.
9
Tracing Neuronal Pathways in Larval Zebrafish
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Tg(lhx5:Kaede)b1204 larvae were anesthetized and mounted laterally in 1.2% low melting point agarose. A small number of cells expressing Kaede were photoconverted from green to red (Ando et al., 2002 (link)) using the 405 nm laser line on a Leica SP8 confocal microscope (objective 25×/0.95NA) by selecting a region of interest (ROI). Mounting the larvae laterally ensured that stray light away from the focal plane would only photoconvert the habenular neuropil. Images of the initial photoconverted region were taken. For both tract-tracing and fate map purposes, larvae were removed from the agarose and incubated for 24 h post conversion, then anesthetized in 0.2% tricaine (MS222, Sigma), placed into Ringer’s solution with tricaine, enucleated and embedded in 0.8% low melt agarose with fish water and imaged on a Leica SP8 confocal microscope.
10
Visualizing Embryonic Cell Dynamics
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To visualize embryonic cells, 10 kDa Alexa 488-dex (total 10 ng) was injected into two blastomeres at the 2-cell stage. To release the CBC pushing force, the tissue around the blastopore was excised, leaving the dorsal side intact. Then, the ventral side of the blastopore slit was cut and embryos were fixed immediately (within 1 min) in 2% TCA and 1.85% formaldehyde. Fixed embryos were dehydrated in methanol and cleared with BABB solution. Fluorescence images were acquired using an SP8 confocal microscope with a 10× objective (Leica).
To examine the CBC shape without incision, 2% TCA and 1.85% formaldehyde were continuously injected into the archenteron at the excretion stage. Fixed and cleared embryos were analyzed using an SP8 confocal microscope with a 10× objective (Leica).
To examine the CBC shape without incision, 2% TCA and 1.85% formaldehyde were continuously injected into the archenteron at the excretion stage. Fixed and cleared embryos were analyzed using an SP8 confocal microscope with a 10× objective (Leica).
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