Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton-100. 5% BSA was used as to block non-specific binding before cells were incubated with primary antibodies overnight at 4 °C (1:500 R&D Systems TuJ1 antibody MAB1195; 1:500 BD α-synuclein antibody 610787; 1:100 Abcam fibrillar synuclein antibody ab209538 and 1:100 ATP synthase subunit-α antibody ab14748; BD MAP2 Alexa488 560399; 1:100 Abcam MTCO1 ab14705; 1:200 Abcam Histone H3 ab1220; 1:100 Abcam TOMM20 Alexa488 ab205486). For ICC, cells were probed with 1:500 Alexa-Fluor 488 (Thermo-Fisher) and 1:500 Alexa-Fluor 594 (Thermo-Fisher) for 1 h at RT. The PLA was carried out using the Duolink in situ red kit (mouse/rabbit; Sigma; DUO92101) according to the manufacturer’s instructions. The mounting media contained DAPI to allow for nuclear staining.
Duolink in situ red kit mouse rabbit
Manufactured by Merck Group
The Duolink In Situ Red Kit Mouse/Rabbit is a laboratory equipment product developed by Merck Group. It is designed to facilitate proximity ligation assays, a technique used to detect and analyze protein-protein interactions within cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Spinning disk confocal microscope, by Leica camera (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Pdia3, by Abcam (1 mentions)
Cellsens software, by Olympus (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
α rabbit plus, by Merck Group (1 mentions)
α mouse minus, by Merck Group (1 mentions)
Lin28a, by Cell Signaling Technology (1 mentions)
Anti upf1, by Cell Signaling Technology (1 mentions)
A1 confocal laser microscope, by Nikon (1 mentions)
Donkey serum, by Jackson ImmunoResearch (1 mentions)
Anti ha tag antibody chip grade, by Abcam (1 mentions)
Vectashield hardset mounting medium with dapi, by Vector Laboratories (1 mentions)
8 protocols using duolink in situ red kit mouse rabbit
1
Multimodal Immunofluorescence and PLA Analysis
2
Proximity Ligation Assay for Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For proximity ligation assay (PLA), the cells were seeded on cover slips and fixed either with (i) 4% formaldehyde for 10 min at 37 °C and then washed twice with 0.02% PBS-T and permeabilized using 0.5% Triton X-100 in PBS for 5 to 10 min at RT or (ii) chilled methanol:acetone 1:1 for 10 min and dried at RT for 3 h (in the case of T47D cell line). Afterward, the cells were incubated with blocking buffer (Sigma–Aldrich—Duolink) for 1 h at 37 °C in a wet chamber. Primary antibodies against AGR2 (1:250; Abnova), PDIA3 (1:500; Abcam), and PDIA6 (1:250; Novus Biologicals) were then incubated in Ab diluent (Duolink) at 4 °C overnight. The PLA was performed with the Duolink In Situ Red Kit Mouse/Rabbit (Sigma–Aldrich) according to the manufacturer's instructions. Anti-mouse MINUS and anti-rabbit PLUS PLA probes (Sigma–Aldrich) were used. Coverslips were mounted with Vectashield mounting medium, and images were acquired by Olympus BX41 microscope using a 40× objective. Images were analyzed with CellSens software (Olympus).
3
Proximity Ligation Assay for Presynaptic Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experiment was performed according to Wang et al. (2015) using the Duolink In Situ Red Kit Mouse/Rabbit (DUO92101; Sigma-Aldrich). Briefly, subsequent to standard dissection and fixation, L3 larvae were blocked for 1 h in PBT containing 5% NGS. Next, samples were incubated with rabbit-α-Cpx (1:500) and mouse-α-Brp (1:250) antibodies overnight at 4°C. After three 10–min washing steps using 0.05% PBT, the samples were incubated for 2 h at 37°C with 1:5 dilution of α-rabbit PLUS (DUO92002; Sigma-Aldrich) and α-mouse MINUS (DUO92004; Sigma-Aldrich) PLA probes, which were added to 5% NGS containing blocking solution. Alexa Fluor 488–conjugated α-HRP antibody (1:250) was added to the mixture in order to enable visualization of neuronal membranes. Following two 5-min washes with Wash buffer A, samples were treated with Ligation solution (1:40 dilution of Ligase in Ligase buffer) for 1 h at 37°C. Again, samples were washed twice for 2 min with Wash buffer A. Next, the samples were incubated in Amplification solution (1:80 dilution of polymerase in amplification buffer) for 2 h at 37°C. After two 10-min washing steps using Wash buffer B, samples were washed twice in 0.01× Wash buffer B and kept in Vectashield-H1000 overnight at 4°C before mounting.
4
In Situ Proximity Ligation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The in situ proximity ligation assay (PLA) was performed on fixed and permeabilised cells using Duolink In Situ Red kit mouse/rabbit (Sigma-Aldrich, DUO92101), according to the manufacturer’s protocol. Briefly, fixed and permeabilised cells were incubated with a blocking solution for 1 h at 37 °C and then incubated with the following primary antibodies: anti-UPF1 (Cell Signaling Technology, 12040; 1:200) and LIN28A (Cell Signaling Technology, 5930; 1:200) overnight at 4 °C. After incubation, the coverslips were washed twice with 1× buffer A, followed by incubation with PLA probes for 1 h at 37 °C. After washing twice with 1× buffer A for 5 min, the coverslip was incubated with the ligation solution for 30 min at 37 °C. Coverslips were washed twice with 1× buffer A, followed by an amplification step with polymerase for 100 min at 37 °C. Finally, the coverslip was washed twice with 1× buffer B and once with 0.01× buffer B and then mounted in VECTASHIELD with DAPI mounting medium (Vector Laboratories, H1200).
5
Proximity Ligation Assay for Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells grown on coverslips to approximately 80% confluence were washed in PBS, fixed in 4% PFA diluted in PBS for 10 min at room temperature, and permeabilized in 0.2% Triton X-100 for 10 min at room temperature. Proximity ligation assays were carried out using Duolink In Situ Red Mouse/Rabbit kit (Sigma), according to the manufacturers’ instructions. Samples were imaged on a Leica Spinning Disk confocal microscope using a 40× lens. Images were analyzed with ImageJ. Antibodies used are in supplemental Table S2 .
6
PLA Assay for Cell Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the PLA assay, 5 × 103 cells were seeded into a confocal dish. Following fixation with 4% paraformaldehyde, the cells were permeabilized, blocked and probed with the indicated antibodies. Afterwards, the Duolink® In Situ Red Mouse/Rabbit kit (DUO92101, Sigma Aldrich) was used to detect the PLA foci according to the manufacturer’s protocol.
7
Proximity Ligation Assay for HMPV-Host Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BEAS-2B cells grown on 10mm coverslips were mock infected or infected with HMPV at an M.O.I. of 2, and 24 h.p.i., cells were fixed with 4% PFA and permeabilized with 1% Triton X-100-PBS. Cells were then incubated in blocking solution at 37°C for 2 hours. A mouse primary antibody for HMPV P and a rabbit β-actin antibody or rabbit α tubulin antibody were added and incubated overnight at 4°C. A proximity ligation assay was then performed using Duolink In Situ red mouse/rabbit kit (Sigma, DUO92101). PLA probes diluted 1:5 were added, and cells were incubated for 1 hour at 37°C in a humidified chamber and processed for ligation for an additional 30 minutes at 37°C. Cells were then washed twice, polymerase was added and DNA was amplified with a florescent substrate for 100 min at 37°C. Coverslips were then mounted on glass slides using Vectashield, and images were taken on a Nikon A1 confocal laser microscope. Images were processed and analyzed for total fluorescent intensity using BlobFinder software.
8
Immunofluorescence for Histone H3.3 G34R
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells grown on coverslips to 80% confluence were washed, fixed in 4% PFA for 10 min, room temperature, and permeabilized (0.2% Triton X-100) for 10 min, room temperature. For mitotracker assays, 100 nM MitoTracker Red CMVRos (Invitrogen) was added for 20 min before fixation. Cells were blocked in 5% donkey serum (Jackson ImmunoResearch) and incubated overnight with primary antibodies Anti-HA tag antibody—ChIP Grade (abcam) and Anti-Histone H3.3 G34R Rabbit Monoclonal Antibody, Clone RM240 (RevMAb BioSciences). After 3 PBS-T washes Secondary antibodies (Fluorescein (FITC) AffiniPure Donkey Anti-Rabbit IgG; Jackson Immuno-Research Laboratories Inc.) were added for 1 h at room temperature. Coverslips were washed 3 times in PBS-T, and mounted with VECTASHIELD® Hardset Mounting Medium with DAPI (Vector Laboratories Inc.). Proximity ligation assays were carried out on coverslips permeabilized as above using Duolink In Situ Red Mouse/Rabbit kit (Sigma). Samples were imaged on a Leica Spinning Disk confocal microscope using a 40X lens. Analysis was carried out with ImageJ.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!