When cells were grown to 80% confluences, 4% paraformaldehyde was added to cells for 30 min. After being blocked by 2.5% normal serum, the podocyte was incubated with anti-p38 (phospho Y182) antibody (Abcam) at 4°C overnight. After washing by PBS, cells were incubated with Alexa Fluor® 594 donkey anti-rabbit IgG (Invitrogen) at room temperature for 2 h. The podocyte was then counterstained by DAPI for 5 min. Cells were observed under a confocal microscope (Leica TCS SP5 MP, Leica, Heidelberg, Germany).
Tcs sp5 mp
Manufactured by Leica
Sourced in Germany
The Leica TCS SP5 MP is a confocal microscope system designed for multi-photon imaging. It offers high-resolution, non-invasive imaging capabilities for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Las af software, by Leica (2 mentions)
Fluorodish 35 mm, by World Precision Instruments (2 mentions)
Spc rabbit antibody, by Abcam (2 mentions)
Anti p38 phospho y182 antibody, by Abcam (1 mentions)
Alexa fluor 594 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit polyclonal to notch1 cleaved val1744 antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions)
Tunel assay kit, by Roche (1 mentions)
Confocal dishes, by NEST Biotechnology (1 mentions)
Sr101, by Merck Group (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Anti rad51, by Abcam (1 mentions)
Dm4000 b led microscope, by Leica (1 mentions)
Dfc310, by Leica (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Tcs sp5 ds, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
38 protocols using tcs sp5 mp
1
Immunofluorescence Analysis of Phosphorylated p38 in Podocytes
2
NF-κB Translocation Assay in U251 Cells
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U251 cells, U251-Wnt1 cells, or U251-EV cells were treated with vehicle, 6-OHDA (50 μM), or/and DKK-1 (100 ng/ml) for 24 h, followed by immunofluorescent staining as described before [27 (link)]. Briefly, cells were washed three times with PBS and fixed in 4% paraformaldehyde-PBS for 15 min at room temperature. The fixed cells were permeabilized with 0.01 M PBS containing 0.1% Triton X-100 for 20 min and then blocked with 10% goat serum for 1 h at room temperature. After blocking, cells were incubated overnight at 4°C with primary antibodies diluted in 10% goat serum/rabbit anti-NF-κB p65 (1 : 800, CST, USA). Cells were washed three times with 0.01 M PBS for 5 min. Alexa Fluor 555-conjugated goat anti-rabbit antibody (CST, USA) was used as a secondary antibody at a dilution of 1 : 1,000 and incubated for 2 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, CST, USA) for 45 s at room temperature before cells were examined under a confocal laser scanning microscope LEICA TCS SP5 MP (Leica, Heidelberg, Germany).
3
Podocyte Immunofluorescence Imaging Protocol
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Podocytes were cultured in a 12-well plate. Upon reaching 80% confluence, the podocytes were treated with different media for 24 h. Then, the podocytes were fixed with 4% paraformaldehyde for 30 min. After blocking with 2.5% dunk serum, the podocytes were incubated with primary antibodies at 4°C overnight. Next, secondary antibodies were added to the cultured podocytes, followed by incubation for 2 h at room temperature. After counterstaining with DAPI, the cells were observed under a confocal microscope (Leica TCS SP5 MP, Leica, Heidelberg, Germany).
4
Immunofluorescence Analysis of Notch1 and Snail
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Cells were cultured on the cover glasses in 24-well plates. When grown to about 80% confluence, cells were fixed with 4% paraformaldehyde for 30 min. After blocking the nonspecific binding sites, cells were incubated with primary antibodies at 4°C overnight. After washing in PBS, cells were incubated with secondary antibodies at room temperature for 2 h, followed by counterstaining with DAPI. Cells were observed under confocal microscope (Leica TCS SP5 MP, Leica, Heidelberg, Germany). Antibodies and dilutions were as follows: rabbit polyclonal to notch1-cleaved-val1744 antibody (1 : 200, Abcam) and goat polyclonal to snail antibody (1 : 200, Everest Biotech).
5
Apoptosis Detection in Cardiovascular Cells
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An in situ cell death detection kit (terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling [TUNEL] assay kit, Roche Applied Science, 11767291910, Basel, Switzerland) was used to assess cell apoptosis in vivo and in vitro. Apoptotic cells in the sections of mouse myocardial tissues were stained according to the manufacturer's instructions, and slides were counterstained for endothelial cells using an antibody against CD31. HUVECs were grown in confocal dishes (NEST, Wuxi, China) before TUNEL staining. The experiment was carried out according to the manufacturer's instructions. 4′,6-diamidino-2-phenylindole (DAPI, Abcam, ab104139, Cambridge, UK) was used to stain the cell nuclei. Images were captured by LEICA TCS SP5 MP (Leica, Weztlar, Germany).
6
Fluorescent Imaging of Cultured Cells
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Cells were cultured on the cover glasses in 24-well plates. When grown to about 80% confluence, cells were fixed with 4% paraformaldehyde for 30 min. After that, cells were incubated with phalloidin for 30 min at room temperature. After washing in PBS, cells were counterstained with DAPI. Cells were observed under a confocal microscope (Leica TCS SP5 MP, Leica, Heidelberg, Germany).
7
Visualizing RBC Morphology in Anticancer Drug Treatment
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An inverted Leica TCS SP5 MP confocal laser microscope (Leica Microsystems GmbH, Wetzlar, Germany) was used to visualize the RBCs’ morphology after incubation with the anticancer drugs. We diluted 10 μL of treated RBCs in 200 μL of HEPES buffer with 3.7% of bovine serum albumin to prevent echinocytosis and placed this sample in Petri dishes (35 mm) with a centre hole replaced by a coverslip (SPL Lifesciences, Pocheon South, Korea). The microphotographs were processed using ImageJ software (Public Domain).
8
Visualizing Astrocytic ChR2 Expression
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At the end of the experiments, SR101 (10 mM in saline, Sigma, Japan), a marker of astrocytes/olligodendrocytes47 (link)48 (link), was intraperitoneally injected (0.10–0.15 mL, N = 6 ChR2 mice). At 3–4 hours after the injection, the brain was removed and fluorescently visualised to verify the expression of ChR2(C128S)-EYFP in SR101-labelled astrocytes using two-photon microscopy (TCS SP5MP, Leica Microsystems)49 (link). Note that ChR2(C128S)-EYFP is expressed throughout the cell membrane including the astrocytic fine processes. The astrocytic expression of ChR2(C128S)-EYFP was dense in the parenchyma of the measured somatomotor cortex, but the astrocytes in the subsurface layers only sparsely expressed the transgene (Fig. 1B ).
9
Immunofluorescence Staining of Cell Markers
10
Upconversion Nanoparticle Cellular Uptake
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The MDA-MB-231 cells were plated over glass coverslips using cell media (DMEM) at 5% CO2 and 37 °C. Cells (7 × 104 cells mL−1) were placed on a fresh coverslip and left to stick for 24 h. Later the cells were cleaned with phosphate buffer solution (PBS) then incubated with UCNC and UCNC@SiO2 (5 µg mL−1 dispersed in DMEM) for 48 h at 5% CO2 and 37 °C. Then 4% paraformaldehyde was used to fix the cells and cell sample was mounted using VECTASHIELD® with DAPI for optical microscopy. TCS SP5 MP (Multi-photon, Leica microsystems), HyD detectors, 100× (HCX PL APO CS 100.0×/1.40 OIL) oil objective and LASAF software (Leica application suite) consisting multi-photon microscopy setup was used for imaging. For excitation, Ti-sapphire femtosecond pulse at 980 nm (1.07 W) was used while performing multi-photon microscopy. The green and red emissions were collected in 520–570 nm and 630–680 nm, respectively.
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