HeLa cells and 501mel melanoma cells were seeded into 8-well chamber CC2-coated slides (Thermo Fisher Scientific) one day before staining. Cells were rinsed with 1X PBS, fixed in 4% paraformaldehyde for 10 minutes, rinsed briefly with 1X PBS 0.1% Tween, then permeabilized with 0.1% Triton for 10 minutes. Following 30 minute block in 1mg/mL BSA (Sigma cat.# A3059), cells were incubated 2 hours with primary antibodies in block (anti-SOX10, Santa Cruz cat.# sc-17342; anti-V5, Invitrogen cat.# 46–0705), then rinsed and incubated 20 minutes in Alexa 488 or 568 secondary antibodies (Invitrogen) diluted in block. Following a 48 hour incubation, cells were fixed and stained to visualize their subcellular localization. Cells were rinsed before mounting with ProLong Gold mounting media with DAPI (Invitrogen). Cell images were taken on Zeiss AxioImager.D2 upright microscope with AxioVision 4.8 software (Carl Zeiss Microscopy, Thornwood, NY).
Alexa 488 or 568 secondary antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 and 568 secondary antibodies are fluorescent dyes that can be conjugated to secondary antibodies. They are used in immunofluorescence techniques to detect and visualize target proteins or antigens in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Axiovision 4, by Zeiss (1 mentions)
Prolong gold mounting media with dapi, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti v5, by Thermo Fisher Scientific (1 mentions)
Anti sox10, by Santa Cruz Biotechnology (1 mentions)
Anti β 3 tubulin, by Cell Signaling Technology (1 mentions)
Eclipse te300 microscope, by Nikon (1 mentions)
Anti sirt2, by Cell Signaling Technology (1 mentions)
2 protocols using alexa 488 or 568 secondary antibodies
1
Immunofluorescent Protein Localization in Cells
2
Immunostaining of EpSCs for SIRT2 and βIII-tubulin
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The culture medium was discarded, and the coverslips from each group were rinsed with PBS (pH 7.4) twice. EpSCs were fixed in 4% paraformaldehyde for 30 min, washed in PBS 3 times, and permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature. After being washed in PBS 3 times, the EpSCs were incubated in PBS-Tween-20 (PBST) with 1% BSA for 60 min to block nonspecific binding of antibodies. Then, the EpSCs were incubated with primary antibody overnight at 4°C. Anti-SIRT2 (Cell Signaling Technology) and anti-βIII-tubulin (Cell Signaling Technology) were used as the primary antibodies. The nuclei were stained with DAPI. After being washed with PBS 3 times, the cultures were incubated with fluorescent Alexa 488 or 568 secondary antibodies (Invitrogen, CA, USA) for 1 h. Immunofluorescence staining was examined using a Nikon Eclipse TE 300 microscope.
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