Tumors isolated from mice were immersed in 4% PFA at 4°C for 2 h, immersed into 30% sucrose solution at 4°C overnight, embedded in tissue fixation solution (OCT), and rapidly frozen at −80°C. The neutrophil (Neu, Santa Cruz Biotech., SC-71674), S. Typhimurium (SL, Bio-Rad, 8,209–4,006), monocyte/macrophage (MOMA-2, Santa Cruz Biotech., SC-59332), and CD206 (M2 macrophage marker, Santa Cruz Biotech., SC-34577) in the tumor tissue sections of mice were analyzed by a standard immunofluorescence staining protocol. Images were obtained under an LSM510 fluorescence microscope (ZEISS, Germany) and processed using laser scanning microscopy (LSM) image software.
Lsm510 fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The LSM510 is a fluorescence microscope manufactured by Zeiss. It is designed for high-resolution imaging of fluorescently labeled samples. The microscope utilizes laser excitation and sensitive detectors to capture detailed images of fluorescent specimens.
Automatically generated - may contain errors
Lab products found in correlation
Oct compound, by Thermo Fisher Scientific (2 mentions)
Moma 2, by Santa Cruz Biotechnology (1 mentions)
2 nbdg, by Cayman Chemical (1 mentions)
Alexa 488 conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti α sma antibody, by Merck Group (1 mentions)
A2547, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Cryomicrotome, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Zeiss (1 mentions)
Anti lc3 antibody, by Abcam (1 mentions)
9 protocols using lsm510 fluorescence microscope
1
Immunofluorescence Analysis of Tumor Infiltrates
2
Immunofluorescence Analysis of AUF1, hnRNP-DL, and RCK/p54
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Commercial HEp-2 cell slides (ANA HEp-2 plus; Generic Assays, Dahlewitz, Germany) were used for immunofluorescence analysis. Slides were incubated with affinity-purified α-AUF1 p45 and α-hnRNP-DL antibodies (undiluted in PBS) or rabbit α-RCK/p54 [37 (link)] antibodies (1:500; University of Florida, Gainesville, Florida, USA) overnight at 4 °C in a moist chamber. After washing, slides were incubated with α-human IgG-FITC antibody (ANA HEp-2 plus; Generic Assays, Dahlewitz, Germany) or polyclonal goat α-rabbit IgG (H+L)-Cy3 (1:50, 111-165-144, Dianova; Hamburg, Germany) antibodies.
HeLa cells were plated and exposed 1 h to 0.5 M sodium arsenite [38 (link)] and afterwards incubated with the respective primary antibodies (affinity-purified α-hnRNP-DL antibody, undiluted in PBS; rabbit α-human AUF1 peptide antibody [19 (link)], 1:1000; mouse α-ATXN2 antibody [39 (link)], 1:200; mouse α-hnRNP-A2/B1, 1:500; Acris Antibodies, San Diego, USA). For immunofluorescence analysis, corresponding FITC- and Cy3-coupled, secondary antibodies were used as previously described [40 (link)].
Preparations were analysed at 400-fold magnification with a LSM510 fluorescence microscope (Carl Zeiss; Jena, Germany) fitted with the appropriate filter sets for FITC and Cy3.
HeLa cells were plated and exposed 1 h to 0.5 M sodium arsenite [38 (link)] and afterwards incubated with the respective primary antibodies (affinity-purified α-hnRNP-DL antibody, undiluted in PBS; rabbit α-human AUF1 peptide antibody [19 (link)], 1:1000; mouse α-ATXN2 antibody [39 (link)], 1:200; mouse α-hnRNP-A2/B1, 1:500; Acris Antibodies, San Diego, USA). For immunofluorescence analysis, corresponding FITC- and Cy3-coupled, secondary antibodies were used as previously described [40 (link)].
Preparations were analysed at 400-fold magnification with a LSM510 fluorescence microscope (Carl Zeiss; Jena, Germany) fitted with the appropriate filter sets for FITC and Cy3.
3
Quantifying Glucose Uptake in Live Cells
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Glucose uptake of the live MPM cells was measured using 2-NBDG as previously described28 (link). Cells are plated in four-well chambered slide for overnight in complete medium. Next day, upon 60% confluency, cells were treated with PFK158 (10 µM, 0–60 min) followed by the incubation with 2-NBDG (150 µg/ml) [Cayman Chemicals, Michigan, USA] for 30 min in the glucose-free medium. Subsequently, cells were washed, mounted, and analyzed in Zeiss LSM510 fluorescence microscope. The fluorescent intensities were calculated using Image J software.
4
Immunofluorescence Staining of α-SMA
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HIFs (1 × 104 cells/well) were seeded onto chamber slides (#30,108, SPL Life Sciences, South Korea) and exposed to TGF-β1 (5 ng/ml) co-incubated with or without TRG/RSG for 24 h, after which the cells were fixed with 4% paraformaldehyde (PFA). After permeabilization with 0.1% Triton-×100 in 1 × PBS and blocking with 5% bovine serum albumin (BSA), the cells were incubated with mouse monoclonal anti-α-SMA antibody (1:500, A2547, Sigma-Aldrich) at 4 °C overnight followed by treatment with goat Alexa488-conjugated anti-mouse IgG antibody (A11029, Thermo Fisher Scientific, Rockford, IL) at room temperature for 2 h. For the staining of actin filaments (F-actin), the cells were incubated with rhodamine-phalloidin (1:200, R415, Invitrogen). After washing, the nuclei were counterstained by Hoechst 33,342 (B2261, Sigma-Aldrich) according to manufacturer’s instructions. The stained sections were immediately covered and examined under a Zeiss LSM510 fluorescence microscope or Zeiss LSM880 confocal laser scanning microscope.
5
Cell Adhesion on Aged Dental Ceramics
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HGFs were cultured for 2 h on G/Z and Y-TZP specimen surfaces before and after aging. After fixation, cell nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Yeasen, Shanghai, Shanghai District, China). Images were obtained with an inverted LSM 510 fluorescence microscope (Carl Zeiss, Jena, Tuttlingen, Germany). The adhered cells were analyzed in randomly selected areas in five sections (450 μm × 450 μm) at a magnification of × 200. The cell adhesion rates were determined through the number of adhered cells divided by the total number of seeded cells.
6
Immunofluorescence Analysis of iNOS in Tumor Tissues
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Mice treated with SL. pNASGFP or PBS (control) were sacrificed on the indicated days after bacterial injection. Tumor tissues were embedded in optimal cutting temperature (OCT) compound (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80 °C. Frozen tumor sections (5 μm thick) were made with a cryomicrotome (Thermo Fisher Scientific, Waltham, MA, USA) and mounted onto glass slides. The slides were blocked with 5 % BSA at room temperature for 1 h, and then incubated overnight at 4 °C with mouse anti-iNOS monoclonal antibody (1:100, Invitrogen, Waltham, MA, USA). After three washes with PBS containing 0.5% Tween-20 (PBS-T), the slides were incubated with Alexa 555-conjugated anti-Rat Ig antibody (1:200, Thermo Fisher Scientific, Waltham, MA, USA) in the dark for 1 h. After washing with PBS-T, the slides were mounted in ProLong antifade mounting solution with 4′,6-diamidino-2-phenylindole dye (DAPI, Invitrogen, Waltham, MA, USA) and sealed with nail polish. The fluorescence signals of sfGFP (green), iNOS (red), and DAPI (blue) were detected using an LSM510 fluorescence microscope (Zeiss, Jena, Germany), and processed using LSM image software.
7
HCMV Infection Inhibition Assay
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HELF cells were pre-plated in a 96-well plate. The following day increasing concentrations of compounds were mixed with HCMV (MOI 0.005) and incubated for 1 h at 37°C. The mixtures were subsequently added to the cells, which were then incubated at 37°C for 3 h; monolayers were then washed and overlaid with 1.2% methylcellulose medium supplemented with 3% FCS and 1mM sodium pyruvate. After five days incubation, cells were observed under an inverted Zeiss LSM510 fluorescence microscope (Zeiss, Oberkochen, Germany) and the percentages of infection were calculated by comparing GFP positive cells in treated and untreated wells.
8
Visualization of HSV-2 Viral Entry
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Cells were plated on coverslips and the following day the binding or entry protocol described above was applied. Cells were than fixed with 4% paraformaldehyde, air dried and for entry protocol permeabilized with PBS-TRITON. Blocking was performed with PBS-BSA 1% for 1 h at room temperature and subsequently HSV-2 polyclonal antibody (1:250) was added on cells, after 1 h at 37°C the inoculum was removed and coverslips were washed with PBS-TWEEN for 3 times. Rhodamine conjugated anti-rabbit (Santa Cruz) was then added on cells for 1 h at 37°C following 3 washes coverslips were mounted on microscope glasses and observed with Zeiss LSM510 fluorescence microscope (Zeiss, Oberkochen, Germany) and images were acquired.
9
Autophagy Visualization in Liver Cancer
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Liver cancer cells were stained with anti-LC3 antibody (abcam; cat. no. ab225383) for 48 h and were then treated with miR-425 inhibitors and stained with 1 µg/ml BODIPY (cat. no. 493/503; Sigma-Aldrich; Merck KGaA) in PBS for 10 min at room temperature. After induction of autophagy by miR-425 inhibitors, the number of mCherry-positive cells was increased compared with that of untreated cells. Red fluorescent protein expression was quantitatively analyzed by ImageJ (v1.52) software. Cells were grown overnight on cover glasses, fixed with 4% paraformaldehyde (37°C and 5% CO2) and sealed with PBS containing 1% bovine serum albumin (BSA; cat. no. ST023; Beyotime Institute of Biotechnology) for 1.5 h at room temperature. Stained samples were visualized using a Zeiss-LSM 510 fluorescence microscope (Zeiss AG; magnification, ×400).
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