For frozen tissue samples, frozen bladder tissue were sectioned and fixed in ice-cold acetone. After blocking with 1% BSA-PBS (Gibco), samples were stained with primary and secondary antibody. For whole mount bladders visualization, bladders were fixed for 2 h in 4 % PFA and blocked with buffer having 0.3 % Trion, 2.5 % normal goat serum (Gibco) in 1% BSA-PBS. Then, samples were stained with primary and secondary antibodies. For imaging human BEC, 5637 cells were grown on glass cover slip and treated as described. 4 % PFA fixed cells and 0.1 % saponin in 1% BSA-PBS solution permeabilized and blocked the samples. Primary antibody and secondary antibodies and phalloidin-Alexa647 (Invitrogen) stained samples. A Nikon ECLIPSE TE200 and Zeiss 780 upright confocal microscope were used for obtaining confocal images via a channel-series approach. For in vitro live imaging, 5637 BECs were grown on MatTek plate (MatTek Corporation) and were infected with UPEC or applied with granules as described. After propidium iodide (Molecular Probes) was applied to the media of cells, cells were placed under live cell station which is maintained at 37°C and 5% CO2 with humidification. Zeiss Axio Observer microscope captured live moments.
Eclipse te200
Manufactured by Zeiss
The Eclipse TE200 is an inverted microscope designed for a variety of research and cell culture applications. It features a sturdy, ergonomic design and provides high-quality optics for bright-field, phase-contrast, and fluorescence imaging. The microscope is equipped with a range of objective lenses and supports various contrast techniques to enable detailed observation and analysis of samples.
Automatically generated - may contain errors
Lab products found in correlation
Apotome, by Zeiss (2 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
780 upright confocal microscope, by Zeiss (1 mentions)
Alexa 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Pbs bsa, by Thermo Fisher Scientific (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
510 meta, by Leica (1 mentions)
Prolong gold containing 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
3 protocols using eclipse te200
1
Immunostaining Protocols for Bladder Tissues
2
Immunocytochemistry of BDNF-treated Cells
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Cultured cells were treated with BDNF for the indicated times, washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were permeabilized with PBS/0.5%Triton X-100 and then blocked for 1 hr with PBS/1% BSA/5% normal goat serum and exposed to primary antibodies overnight at 4°C. Cells were washed 3X with PBS, and exposed to secondary antibodies coupled to different fluorophores for 1 hr at room temp. Cells were washed three times in PBS and then mounted using Prolong Gold containing 4′,6′-diamidino-2-phenylindole (DAPI) (Life Technologies P36934). Cells were analyzed by epifluorescence (Nikon Eclipse TE200), confocal (Zeiss 510 Meta), or enhanced resolution (Leica SP8, 63X, 1.4 NA, Lightning Mode) microscopy. No immunostaining was seen in controls with omission of the primary antibodies.
3
Immunofluorescence Staining of Cells
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Cells were fixed in 4% (wt/vol) paraformaldehyde in PBS (Sigma), washed three times in PBS and permeabilized with 0.1% (vol/vol) Triton X-100 (Sigma) in PBS. Samples were treated with blocking solution containing 5% (vol/vol) BSA and 5% (vol/vol) not immuno serum in PBS. Primary antibodies were incubated over-night at +4°C and their working dilutions are listed in Table 2. Cells were incubated with suitable secondary antibodies (Alexa Fluor) for 45 min. Nuclei were stained with 4',6-diamidino-2phenylindole (DAPI, Sigma). Samples were observed under a Nikon Eclipse TE200 and Zeiss Apotome.
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