HeLa cells grown on sterile glass coverslips were washed with ice-cold PBS and fixed with 4% PFA in PBS for 20 min at RT. Then, cells were permeabilised using 0.1% Triton X-100 in PBS for 5 min and blocked for 15 min using PBS supplemented with 1% BSA. Cells were labelled with primary antibodies diluted in blocking buffer at RT for 1 h, washed and labelled with mouse PLUS and Rabbit MINUS antibodies for 1 h at 37 °C. The PLA signal was visualised using the in situ PLA detection kit (Sigma) according to the manufacturer’s protocol. After labelling the cells were mounted using Prolong Gold antifade reagent with DAPI and imaged on a DeltaVision widefield microscope using a ×100/1.4 A immersion objective. Pictures were deconvolved using Softworx software and analysed using Fiji ImageJ v1.48q.
In situ pla detection kit
Manufactured by Merck Group
The In situ PLA detection kit is a laboratory tool used to visualize and analyze protein-protein interactions within cells. It employs a proximity ligation assay (PLA) technique to detect and localize specific protein complexes in their native cellular environment. The kit provides the necessary reagents and protocols to perform this analysis.
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Lab products found in correlation
3 protocols using in situ pla detection kit
1
Immunofluorescence and In Situ PLA Assay
2
In situ Proximity Ligation Assay
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Cells were washed 3 times in PB and incubated in mouse PLUS and rabbit MINUS probes (Sigma Aldrich) according to the manufacturer’s protocol. Cells were washed in PB before detection using the in situ PLA detection kit (Sigma-Aldrich) as previously described (van Loosdregt et al., 2011 (link)). Samples were analyzed with a 63x objective on a Zeiss LSM 710 microscope (Carl Zeiss).
3
In Situ Proximity Ligation Assay
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Cells were washed 3 times in PB and incubated in mouse PLUS and rabbit MINUS probes (Sigma Aldrich) according to the manufacturer's protocol. Cells were washed in PB before detection using the in situ PLA detection kit (Sigma-Aldrich) as previously described 37 . Samples were analyzed with a 63x objective on a Leica SP8X.
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