Protocol - Labelling Lysosomes in live cells
Labelling Lysosomes in HaCAT cells after UVB stimulation
Category:
In situ hybridization and Immunochemistry
Last revision: Jan 29, 2015
Author(s):
A. Kisslinger, S.Paladino,D.Tramontano
Contact
Name: Kisslinger Annamaria
Address: Institute of Experimental Endocrinology and Oncology “G. Salvatore” , CNR via Pansini 5, 80131 Naples Italy
Phone: +3908107464552
Email: a.kisslinger@ieos.cnr.it
Figure legend:
Steps
Description | Temperature | Time | Note |
---|---|---|---|
Seed cells on coverslips
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37°C,5% CO2 in a humidified incubator
|
24 hrs before experiment
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Experimental culture conditions: semiconfluent
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Incubated with Lysotracker Probe Molecular Probes)
|
37°C,5% CO2 in a humidified incubator
|
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)
|
Wash with PBS Buffer
|
RT
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three times
|
|
Fix with Paraformaldheyde (PFA) 4%
|
RT
|
20 min
|
|
Wash with PBS Buffer
|
RT
|
three times
|
|
Quench with 50 mM NH4Cl
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RT
|
15 min
|
|
Permeabilized with PBS-0.2%Triton X-100
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RT
|
5 min
|
|
Wash with 1% BSA/PBS
|
RT
|
1x5min
|
|
Incubate with 0,5%BSA/PBS
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RT
|
20-30 min
|
|
Add primary antibody alpha-tubulin
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Humidified chambr
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20 min
|
1:500
|
Wash with 0,5% BSA/PBS
|
RT
|
3x5 min
|
|
Add FITC-conjugate secondary antibodies
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Humidified chamber
|
20 min
|
1:1000 antimouse
|
Wash with 0,5% BSA/PBS
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RT
|
3 X 10 min
|
|
Add nuclear fluorescent stain DAPI
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RT
|
10 min
|
1:1000 PBS
|
Wash with 0,5% BSA/PBS Buffer
|
RT
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3 x 5 min
|
|
Place a small drop of mounting medium on the microscope slide
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1:1 PBS Glycerol
|
||
Slowly lower the cover glass onto the mounting medium keep attention to disturbing bubbles
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pay attention to disturbing bubbles
|
||
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
Quality validation:
Yes
Validation info
The protocol has been used to obtain data published in a peer-reviewed journal.