Leica sp5 confocal microscope

Manufactured by Leica Microsystems

Sourced in Germany, United Kingdom, United States, Australia

The Leica SP5 is a confocal microscope designed for advanced microscopy applications. It features a modular design, allowing for customization to meet specific research needs. The SP5 provides high-resolution imaging and supports a range of sample types and microscopy techniques.

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198 protocols using leica sp5 confocal microscope

Measuring ROS in 16HBE cells

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ROS generation in 16HBE cells was measured using the oxidant-sensitive fluorometric probe DCFH-DA (cat. no. S0033; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. In brief, the cells were cultured in the plate on glass slides and subjected to different treatments as follows: i) Control group; ii), HDM group; iii) RES group; iv) HDM combined with RES group; v) NAC group; and vi) HDM combined with NAC group. After treatment, cells were washed with PBS and then incubated with 10 µM DCFH-DA in DMEM for 30 min at 37°C. The cells were then washed with PBS and images were captured using a fluorescence microscope (SP5 Leica confocal microscope; Leica Microsystems GmbH).
Frozen lung tissues were incubated with 25 µM dihydroethidium (DHE) (cat. no. BB-470515, BestBio) in PBS for 15 min at 37°C. The sections were washed with PBS for 3 min and then imaged using a fluorescence microscope (SP5 Leica confocal microscope; Leica Microsystems GmbH).

Zhang Y., Guo L., Law B.Y., Liang X., Ma N., Xu G., Wang X., Yuan X., Tang H., Chen Q., Wong V.K, & Wang X. (2020). Resveratrol decreases cell apoptosis through inhibiting DNA damage in bronchial epithelial cells. International Journal of Molecular Medicine, 45(6), 1673-1684.

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Mannitol-Induced Membrane Dynamics in Plants

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Four-day-old seedlings were incubated in 1/2 MS medium (CK), 1/2 MS medium supplemented with mannitol (0.15 M and 0.3 M final concentration) together with 3 μM FM4-64 (Merck KGaA, Darmstadt, Germany) for 5 min at room temperature and then observed under a Leica SP5 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany).
For the inhibitor treatments, 4-day-old seedlings were incubated in MβCD (Merck KGaA, Darmstadt, Germany) (diluted to 10 mM with 1/2 MS medium) or TyrA23 (Merck KGaA, Darmstadt, Germany) (diluted to 50 μM with 1/2 MS medium) for 30 min, followed by incubation for an additional 5 min by the addition of 3 μM FM4-64 (final concentration) in the absence or presence of mannitol (0.15 M or 0.3 M final concentration). After rinsing with the same solution (without FM4-64) for 1 min, the seedlings were observed under a Leica SP5 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) fitted with a 63× oil objective, excited using 488 nm wavelength for GFP and 561 nm wavelength for FM4-64 respectively.

Wu Z., Fan C., Man Y., Zhang Y., Li R., Li X, & Jing Y. (2021). Both Clathrin-Mediated and Membrane Microdomain-Associated Endocytosis Contribute to the Cellular Adaptation to Hyperosmotic Stress in Arabidopsis. International Journal of Molecular Sciences, 22(22), 12534.

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Quantitative Analysis of Organoid Size

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Organoid size was analyzed on days 25, 50 and 90 for in vitro organoids. Fixed, cryopreserved and cryosectioned organoids (12–15 μm sections) were imaged following nuclear staining using Hoechst using a Leica Confocal SP5 microscope (Leica Microsystems, Wetzlar, Germany) with an air 4× objective driven by Las-AF software. Image J/Fiji software was used to evaluate the area of the organoid pictures and represented as mm².
For day 25 and day 90 PC1 (n = 4 organoids), PC2 (n = 4 organoids) and PC3 (n = 4 organoids), statistical analysis was performed using a two-way ANOVA with Tukey post-tests; ns = non-significant.

Magni M., Bossi B., Conforti P., Galimberti M., Dezi F., Lischetti T., He X., Barker R.A., Zuccato C., Espuny-Camacho I, & Cattaneo E. (2022). Brain Regional Identity and Cell Type Specificity Landscape of Human Cortical Organoid Models. International Journal of Molecular Sciences, 23(21), 13159.

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Immunofluorescence Imaging of MDCK Monolayers

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After TER measurements, MDCK monolayers were washed three times with ice-cold PBS/Ca²⁺, fixed with 4% paraformaldehyde for 30 min at 4°C, permeabilized with 0.1% Triton X-100 for 5 min, blocked for 30 min with 3% BSA and treated for 1 h at 37°C with a specific primary antibody. The monolayers were then rinsed 3 times with PBS/Ca²⁺, incubated with an appropriate FITC or TRICT-labeled antibodies for 30 min at room temperature and rinsed as indicated above. Filters with the cells were excised with a scalpel and mounted in Vectashield (Vector Labs, Burlingame, CA, USA). The preparations were examined with a Leica confocal SP5 microscope (Leica Microsystems, Wetzlar, Germany). The captured images were imported into FIJI, version 2.8 (National Institutes of Health, Baltimore, USA), to obtain maximum projections and into the GNU Image Manipulation Program (GIMP) to normalize brightness and contrast in all images and construct figures.

Rocha S.C., Pessoa M.T., Neves L.D., Alves S.L., Silva L.M., Santos H.L., Oliveira S.M., Taranto A.G., Comar M., Gomes I.V., Santos F.V., Paixão N., Quintas L.E., Noël F., Pereira A.F., Tessis A.C., Gomes N.L., Moreira O.C., Rincon-Heredia R., Varotti F.P., Blanco G., Villar J.A., Contreras R.G, & Barbosa L.A. (2014). 21-Benzylidene Digoxin: A Proapoptotic Cardenolide of Cancer Cells That Up-Regulates Na,K-ATPase and Epithelial Tight Junctions. PLoS ONE, 9(10), e108776.

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Labeling and Imaging of NAc Neurons

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Neurons from nucleus accumbens (NAc) were labeled using the Helios Gene Gun System (Bio-Rad, Hercules, CA) as previously described (23) . DiI-labeled medium spiny neurons of the NAc were imaged using a Leica Confocal SP5 microscope (Leica Microsystems, Wetzlar, Germany) with a 363 oilimmersion objective. Details for the imaging conditions are described in the Supplement.

Brito V., Giralt A., Masana M., Royes A., Espina M., Sieiro E., Alberch J., Castañé A., Girault J.A, & Ginés S. (2019). Cyclin-Dependent Kinase 5 Dysfunction Contributes to Depressive-like Behaviors in Huntington's Disease by Altering the DARPP-32 Phosphorylation Status in the Nucleus Accumbens. Biological psychiatry, 86(3).

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Lyophilized DNV Viability Assessment

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Samples of DNVs loaded with AF647-Zol produced by the modified microfluidic method were dialyzed and lyophilized as described below. The lyophilized samples were then resuspended to a 100x dilution of their original postsynthesis concentration. Resuspended samples were imaged in fluid on a glass slide through an SP5 Leica Confocal Microscope (Leica Microsystems, Waltzer, Germany) to determine postlyophilization vesicle viability and drug leakage.

Subbiah N., Campagna J., Spilman P., Alam M.P., Sharma S., Hokugo A., Nishimura I, & John V. (2017). Deformable Nanovesicles Synthesized through an Adaptable Microfluidic Platform for Enhanced Localized Transdermal Drug Delivery. Journal of Drug Delivery, 2017, 4759839.

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Immunofluorescence Analysis of hnRNP H in Spinal Cord

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Spinal cord sections were acquired from the Columbia Brain Bank and embedded in optimum cutting temperature (O.C.T.) compound (Sakura, Torrance, CA) and frozen at −80°C. Consecutive sections (25μ thick) were cut using a freezing microtome (Leica CM 3050S), mounted onto microscope slides, and stored at −80°C. Prior to fixation sections were briefly thawed and rinsed with PBS, then fixed in 4% paraformaldehyde in PBS for 15 min blocked and washed three times with PBS for 5 min each. Sections were blocked with 5% normal donkey serum diluted in Tris buffered saline (pH 7.4) with 0.2% Triton X-100 (TBS-T) and incubated with hnRNP H antibody (Bethyl) diluted in TBS-T with 5% normal donkey serum overnight at 4°C. After washing with TBS-T, tissue sections were incubated for 4 hrs at RT with species-specific secondary antibody coupled to Alexa 568 (1:1000; Life Technologies, Carlsbad, CA). After washing with TBS-T, coverslips were applied with Flouromount G (Southern Biotech, Birmingham, AL) and imaged in a blinded fashion using an SP5 Leica confocal microscope (Leica Microsystems, Wetzlar, Germany). The images were converted into z-stack max projections and individual foci were measured using ImageJ.

Conlon E.G., Lu L., Sharma A., Yamazaki T., Tang T., Shneider N.A, & Manley J.L. (2016). The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains. eLife, 5, e17820.

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Bronchial Epithelial Cell Response to HDM and Resveratrol

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The bronchial epithelial cells (1×10⁵ cells) were cultured in the plate on glass slides and cells were subjected to different treatments as follows: i) Control group; ii) HDM group; and iii) HDM combined with RES group. Following treatment, cells were fixed with ice-cold methanol for 10 min at room temperature and incubated with primary antibody targeting γH2AX (1:200; cat. no. 05-636; EMD Millipore) and 8-OHdG (1:200; cat. no. ab48508; Abcam) at room temperature for 2 h, followed by Alexa Fluor 555 conjugated secondary antibody (1:500; cat. no. A32727; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. The nuclei were stained with DAPI at room temperature for 5 min. Following staining, the differences were observed by using SP5 Leica confocal microscope with Leica Application Suite Software (version no. 14.0.0.162; Leica Microsystems GmbH).

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Quantifying Apoptosis in Lung Tissues

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Lung tissues were harvested and fixed with 10% formalin for 24 h at room temperature, and then embedded in paraffin and cut into 5-µm sections. The tissue sections were deparaffinized and rehydrated, and then treated with citric acid for antigen retrieval for 10 min at room temperature. The TUNEL Assay Apoptosis Detection kit (cat. no. C1088; Beyotime Institute of Biotechnology) was used to detect DNA fragmentation. In brief, the tissue sections were treated with formaldehyde on ice for 15 min. Subsequently, the tissue sections were washed with phosphate-buffered saline (PBS) and 70% ice-cold ethanol was added followed by incubation for 30 min. The tissue sections were again washed with PBS three times for 5 min each time, staining solution was added and the sections were incubated at 37°C for 60 min. The tissue sections were then washed and treated with ribonuclease A (RNase A) for 30 min at room temperature, then visualized under a fluorescence microscope (SP5 Leica confocal microscope; Leica Microsystems GmbH). The number of TUNEL-positive cells were counted in five different fields for each stained section.

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Immunofluorescence Analysis of Lung Tissue Damage

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For immunofluorescence (IF) analysis, the sections of lung tissues were fixed with 4% formaldehyde and permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature. Subsequently, the slides were incubated with 2% serum-blocking buffer for 20 min at room temperature and then incubated with specific antibodies. The primary antibodies against γH2AX (1:200; cat. no. 05-636, EMD Millipore) and 8-OHdG (1:200; cat. no. ab48508, Abcam) were added and incubated at room temperature for 2 h, followed by incubation with the Alexa Fluor 555 conjugated secondary antibody (1:500; cat. no. A32727; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h. The nuclei were stained with DAPI (cat. no. C1005; Beyotime Institute of Biotechnology) at room temperature for 5 min. Differences in immunostaining were detected by using SP5 Leica confocal microscope with Leica Application Suite Software (version 14.0.0.162, Leica Microsystems GmbH).

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