Immunofluorescence (IF) was performed as described previously (25 (link)). Briefly, cells were cultured on pre-coated glass coverslips for IF or glass-bottom plates for live-cell imaging containing normal cell culture medium before analysis. Cells were washed with PBS three times and pre-extracted with CSK buffer (10 mM PIPES pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100) for 5 min on ice followed by 15 min fixation with 2% paraformaldehyde in PBS at room temperature. Samples were washed with PBS three times and then incubated with the indicated antibodies overnight at 4°C. On the following day, samples were washed with PBS three times, and incubated with secondary antibodies for 1 h at room temperature. Samples were then mounted with VECTASHIELD mounting solution containing DAPI (Vector Labs). Samples were visualized using an inverted FV3000 scanning confocal microscope (Olympus). Z-stacked images were obtained for images and focus counting, which was performed with FW31S software.
Vectashield mounting solution containing dapi
Manufactured by Vector Laboratories
Vectashield mounting solution containing DAPI is a fluid medium designed to be used for mounting and preserving biological samples on microscope slides. It contains the fluorescent dye DAPI, which binds to DNA and emits blue fluorescence when excited by ultraviolet light. This product is intended for use in fluorescence microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Inverted fv3000 scanning confocal microscope, by Olympus (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Fluorescence conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Axiovs40, by Zeiss (1 mentions)
Dfc340 fx camera, by Leica (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ctr 5500 microscope, by Leica (1 mentions)
Lsm 510, by Zeiss (1 mentions)
4 protocols using vectashield mounting solution containing dapi
1
Immunofluorescence Microscopy Protocol
2
Immunofluorescence Analysis of Pancreatic Progenitors
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Co-cultured or control mMSCs grown on coverslips (BD BioCoat™) were fixed with 4% paraformaldehyde for 20 minutes. After fixation, the cells were washed, permeabilized, and blocked in phosphate-buffered saline (PBS) containing 0.2% Triton X-100 and 5% FBS for 30 minutes at room temperature. Cells were then incubated with goat polyclonal anti-Ptf1α (1: 200), rabbit polyclonal anti-Mist-1 (1: 200) in PBS containing 0.1% Triton X-100 and 1% FBS at 4°C overnight. After washing with PBS, the cells were incubated at room temperature for 1 h with appropriate fluorescence-conjugated secondary antibodies (1: 200 dilution, Molecular Probes). Coverslips were mounted and nuclei stained with Vectashield mounting solution containing DAPI (Vector Laboratories Ltd). Fluorescence was observed under a 100X magnification using a Zeiss Axiovert 200M microscope equipped with a Zeiss AxioCam MRm camera and images were obtained from AxioVs40 software (Ver. 4.7.1.0, Zeiss).
3
Immunofluorescence Analysis of Patellar Tendons
4
Metastasis Modeling in Nude Mice
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Nude mice were randomly divided into four groups each group has 10 mice, 5 mice for 2 weeks model study and 5 mice for 3days model study for the administration of DU145 cells. Cells (0.5×10 6 ) suspended in sterile normal saline were injected (i.v.) to each group of mice separately via the tail vein.In all experiments, mice were assessed for metastasis at 3 days and 2 weeks after DU145 cells were administered. 3 days model designed for detecting early metastasis stage and 2 weeks model for late stage. For 2 weeks mouse model, 1.5 ml of 15 % India-ink solution was injected intratracheally to stain the lungs and visualize non-stained the tumor nodules. Then lung were then harvested and put in Fekete"s solution(300 ml 70 % ethanol, 30 ml 37 % formaldehyde, 5 ml glacial acetic acid), then put in fresh Fekete's solution overnight. The metastasis tumor nodules are white. For 3 days mouse model, mice lung tissue was made into frozen sections, then sections mounted with coverslips with Vectashield mounting solution containing DAPI (Vector Laboratories, Burlingame, CA). The number of GFP containing cancer cells was viewed under confocal imaging microscope (LSM510, Carl Zeiss, Germany).
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