Protocol - Labelling Lysosomes in live cells
Labelling Lysosomes in HaCAT cells after UVB stimulation
Figure legend:
HaCaT cells, grown on coverslips, were loaded with lysotracker (in red) A, fixed and stained with a specific antibody against tubulin and revealed by FITC-conjugated secondary antibodies (green) B. Nuclei were labeled with DAPI (blue) C. Overlapping of ABC. Serial confocal sections were collected. Bar, 8 µm.
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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Seed cells on coverslips
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37°C,5% CO2 in a humidified incubator
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24 hrs before experiment
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Experimental culture conditions: semiconfluent
|
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Incubated with Lysotracker Probe Molecular Probes)
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37°C,5% CO2 in a humidified incubator
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1 hrs after UVB stimulation
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In culture medium (Dulbecco Modified Eagle's Medium (DMEM) , 1%FBS with 1:500/1:1000 stock solution as furnished by Molecular Probes)
|
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Wash with PBS Buffer
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RT
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three times
|
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Fix with Paraformaldheyde (PFA) 4%
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RT
|
20 min
|
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Wash with PBS Buffer
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RT
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three times
|
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Quench with 50 mM NH4Cl
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RT
|
15 min
|
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Permeabilized with PBS-0.2%Triton X-100
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RT
|
5 min
|
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Wash with 1% BSA/PBS
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RT
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1x5min
|
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Incubate with 0,5%BSA/PBS
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RT
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20-30 min
|
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Add primary antibody alpha-tubulin
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Humidified chambr
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20 min
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1:500
|
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Wash with 0,5% BSA/PBS
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RT
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3x5 min
|
|
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Add FITC-conjugate secondary antibodies
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Humidified chamber
|
20 min
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1:1000 antimouse
|
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Wash with 0,5% BSA/PBS
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RT
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3 X 10 min
|
|
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Add nuclear fluorescent stain DAPI
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RT
|
10 min
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1:1000 PBS
|
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Wash with 0,5% BSA/PBS Buffer
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RT
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3 x 5 min
|
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Place a small drop of mounting medium on the microscope slide
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1:1 PBS Glycerol
|
||
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Slowly lower the cover glass onto the mounting medium keep attention to disturbing bubbles
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pay attention to disturbing bubbles
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Collect image
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Other informations
The irradiating source consists of three lamps (Philips Ultraviolet 8 TL 20W/01 RS lamps; Philips, Eindhoven, Netherlands) generating UVB light in the range of 290–320 nm with an emission peak at 312 nm. Intensity of UVB irradiation was measured using a phototherapy radiometer (International Light, Newburyport, MA).
Images were collected using a laser scanning microscope (LSM 510 META, Carl Zeiss Microimaging, Inc.) equipped with a planapo 63x oil-immersion (NA 1.4) objective lens. All image processing was done using LSM 510 software
Validation info
The protocol has been used to obtain data published in a peer-reviewed journal.
