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Home  |  Protocols  |  In situ hybridization and Immunochemistry  |  Labelling Lysosomes in live cells

Protocol - Labelling Lysosomes in live cells

Labelling Lysosomes in HaCAT cells after UVB stimulation

CategoryIn situ hybridization and Immunochemistry
Last revisionJan 29, 2015
Author(s)A. Kisslinger, S.Paladino,D.Tramontano
NameKisslinger Annamaria
AddressInstitute of Experimental Endocrinology and Oncology “G. Salvatore” , CNR via Pansini 5, 80131 Naples Italy


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.


Description Temperature Time Note
Seed cells on coverslips
37°C,5% CO2 in a humidified incubator
24 hrs before experiment
Experimental culture conditions: semiconfluent
Incubated with Lysotracker Probe Molecular Probes)
37°C,5% CO2 in a humidified incubator
1 hrs after UVB stimulation
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
three times
Fix with Paraformaldheyde (PFA) 4%
20 min
Wash with PBS Buffer
three times
Quench with 50 mM NH4Cl
15 min
Permeabilized with PBS-0.2%Triton X-100
5 min
Wash with 1% BSA/PBS
Incubate with 0,5%BSA/PBS
20-30 min
Add primary antibody alpha-tubulin
Humidified chambr
20 min
Wash with 0,5% BSA/PBS
3x5 min
Add FITC-conjugate secondary antibodies
Humidified chamber
20 min
1:1000 antimouse
Wash with 0,5% BSA/PBS
3 X 10 min
Add nuclear fluorescent stain DAPI
10 min
1:1000 PBS
Wash with 0,5% BSA/PBS Buffer
3 x 5 min
Place a small drop of mounting medium on the microscope slide
1:1 PBS Glycerol
Slowly lower the cover glass onto the mounting medium keep attention to disturbing bubbles
pay attention to disturbing bubbles
Collect image

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.


Vitale N, Kisslinger A, Paladino S, Procaccini C, Matarese G, Pierantoni GM, Mancini FP, Tramontano D. Resveratrol couples apoptosis with autophagy in UVB-irradiated HaCaT cells PLoS One. 2013;8,e80728.


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