Duolink® PLA Fluorescence Protocol This protocol describes the immunofluorescence detection, visualization and quantification of individual proteins, protein modifications, and protein interactions in tissue and cell samples.

Duolink® PLA Fluorescence Protocol

This protocol describes the immunofluorescence detection, visualization and quantification of individual protein , protein modification and protein interactions in tissue and cell samples. To learn more about Duolink® PLA technology, download our manual and review how to optimize Duolink® adjacency connection assays for more details. For other experimental protocols, see the Duolink® PLA brightfield experimental protocol and the Duolink® PLA Probemaker User Guide. For more content, please visit Merck Life Sciences official website: www.sigmaaldrich.cn

Materials and equipment

Duolink

For specific product suggestions, please refer to the Duolink® PLA product selection guide. Simply put, to run a Duolink® PLP experiment for fluorescence detection , the following Duolink® PLA products are required:

1. Duolink® PLA probe (a PLUS and a MINUS from different species that match your primary antibody host species). Each kit includes: Note: Store all components of the kit at 4°C.

   Note: If required, custom PLA probes can be generated using the Duolink® PLA Probemaker PLUS and/or Probemaker MINUS kit. For more information, see the Probemaker guide.

1. PLA Probe (5x)— PLUS and/or MINUS, depending on the product

2. Blocking Solution—for blocking samples before incubating antibodies

3. Antibody Diluent—for diluting the PLA probe and, if required, for diluting primary antibody

2. Duolink® fluorescence detection reagent (green, orange, red and far red are available). Each kit includes: Note: Store all components at -20°C.

2. ligation stock solution (5x) – Dilute to prepare 1x ligation buffer

3. ligase (1 U/μL) – Add to prepare ligation solution

4. amplification stock solution (5x) – Dilute to prepare 1x ligation buffer

5. Polymerase (10 U/μL) - Add to prepare amplification solution

3. fluorescence with wash buffer (A and B)

4. DAPI-containing Duolink® mounting agent (for slide , stored at 2-8°C) or nuclear staining and anti-fade agent (for microplate, stored at -20°C)

Other materials

1. primary antibody for detecting target proteins. When used with Duolink® PLA probes, they must be raised in mice, rabbits, or goats.

2. High purity Pure water (sterile filtering, Milli-Q® or similar water quality)

3. Samples (cells or tissues) on slides have been pretreated in fixation, recovery and permeabilization. For the relevant suggestions and details on the following topics, see How to Optimize Duolink® Proximity Connection Assay:

2. Experimental Design and Control

3. Select primary antibody and Optimize

4. Sample Processing

Device

1. Fluorescence Microscope , with appropriate filters, cameras and for image acquisition Software

2. 37°C incubator

3. shaker

4. heating humidity box or microplate heat transfer seat

5. hydrophobic pen, used to draw reaction area

6. freezer seat (for enzymes)

7. staining jar

8. tweezers

9. pipette and suction head (1 μl to 1000 μl)

10. coverslips compatible with fluorescence microscope

Duolink

2Duolink

2 The following experimental protocol is suitable for 1cm2 samples on slides, and 40 μL of solution is required to cover them fully. Adjust volume according to your reaction area and sample quantity. All incubations should be performed in a humidity chamber. All washing steps should be performed at room temperature in a staining jar with at least 70 ml buffer in the jar and stir gently.

Preparation reagent

1. Washing buffer A and B should be prepared before starting the assay, dissolve a small bag of contents in high-purity pure water, and set a volume of 1000 mL. The solution can be stored at room temperature for a short time (less than two weeks) or at 4°C for a long time. Note: Warm the solution to room temperature before use. Before starting the assay,

2. , dilute 1x wash buffer B with 1:100 with high purity water to prepare 0.01x wash buffer B.

3. Many Duolink® PLA reagents are provided in concentrate stock form and are diluted immediately before use. Do not store diluted Duolink® PLA reagent.

Duolink

Before starting, samples were deposited on slides and pretreated in fixation, recovery and/or permeabilization. For more information, see How to Optimize Duolink® Proximity Connection Assays.

1. blocking

1. Vortex Duolink® blocking solution.

2. Add 1 drop (~40 μL) of Duolink® blocking solution to each 1cm2 sample. Make sure the blocking solution covers the entire sample.

3. Place the slides in a heating humidity chamber at 37°C for 60 minutes.

2. Incubate primary antibody Do not let the slides dry before adding the antibody, otherwise it will cause background.

1. Vortex Duolink® antibody diluent.

2. dilute primary antibody or antibody to appropriate concentration in Duolink® antibody diluent.

3. ejected Duolink® blocking liquid

4. added primary antibody solution to each sample.

5. Incubate the slides in a humidity chamber. Incubate the primary antibody with the optimal incubation temperature and time.

3. Incubate the Duolink® PLA probe
   

1. Vortex PLUS and MINUS PLA probe

2. dilute the PLUS and MINUS PLA probes in Duolink® antibody diluent with 1:5.
   For 40 μL reaction, take 8 μL of PLA probe MINUS stock solution, 8 μL of PLA probe PLUS stock solution and 24 μL of antibody diluent. Prepare sufficient solution for all samples.

3. Remove primary antibody solution from slides

4. Wash slides in 1x wash buffer A for 2x 5 minutes at room temperature.

5. Remove excess washing buffer and apply PLA probe solution.

6. The slides were placed in a preheated humidity chamber at 37°C for 1 hour.

4. Connecting
   Instructions: The ligase must be added immediately when it needs to be added to the sample. Make sure the ligation buffer is completely thawed and mixed thoroughly before use.

1. dilute the 5x Duolink® ligation buffer with 1:5 with high purity water and mix.
   For 40 μL reaction, add 8 μL 5x ligation buffer to 32 μL high-purity pure water. Prepare sufficient solution for all samples.

2. Remove the PLA probe solution from the slide.

3. Wash slides in 1x wash buffer A for 2x 5 minutes at room temperature.

4. During the washing process, the ligase removed from the freezer was recovered using a freezer (-20°C).

5. added ligase to the 1x ligation buffer in step (a) at a 1:40 dilution and mixed.
   For 40 μL of ligation solution, add 1 μL of ligase to 39 μL of 1x ligation buffer.

6. Remove excess washing buffer and then apply the ligation solution.

7. Place the slides in a preheated humidity chamber at 37°C for 30 minutes.

5. amplification
   instructions: Polymerase should be added immediately when it needs to be added to the sample. Amplification buffer is light sensitive. Avoid light exposure to all solutions containing amplification buffer.

1. 5x amplification buffer was diluted 1:5 with high purity water and mixed. For 40 μL of reaction, add 8 μL of 5x amplification buffer to 32 μL of high purity pure water. Prepare sufficient solution for all samples.

2. removes the ligation solution from the slide.

3. Wash slides in 1x wash buffer A for 2x 5 minutes at room temperature.

4. During the washing process, the polymerase removed from the freezer was recovered using a freezer (-20°C).

5. Add polymerase to the 1x amplification buffer in step (a) at a 1:80 dilution and mix.

6. Remove excess washing buffer and then apply the amplification solution.

7. Place the slides in a preheated humidity chamber at 37°C for 100 minutes.

6. Final washing
   Instructions: Photosensitive reagent. Always avoid preserving slides.

1. removes the amplification solution from the slide.

2. Wash slides in 1x wash buffer B for 2x 10 minutes at room temperature.

3. Wash slides in 0.01x wash buffer B for 1 minute.

7. Preparation for imaging
   Instructions: Duolink® in situ sealant containing DAPI is water-based and does not solidify. The edges of the coverslip can be sealed onto the slide using clear nail polish. Avoid air bubbles entangled under the coverslip.

1. Remove excess washing buffer from the slides.

2. Use the minimum volume of Duolink® in situ mounting medium containing DAPI and cover the slide with a coverslip.

3. wait for 15 minutes and then analyze in a fluorescence or confocal microscope using at least 20x objective lens. After imaging of

4. , the slides can be stored at 4°C in the dark for up to 4 days, or at -20°C for up to 6 months

results

Image acquisition results of

Duolink® PLA experiments are usually observed using fluorescence microscopes and fluorophores are detected with appropriate filters. The Duolink® PLA signal is considered to be discrete fluorescent spots at different locations of the cells studied (see Figure 1A). A single signal has a submicron size and can be in multiple focal planes. Therefore, it may be necessary to obtain images over the entire sample thickness. However, as long as all images to be compared come from similar positions within the sample, images can be acquired in only one plane. It is worth noting that some signals are often detected in technical negative controls (for example, controls without primary antibodies, see Figure 1B). However, if more than one or two signals are obtained per ten cells, further titration of primary antibodies may be required.

It is important to use the same settings (exposure time, gain, filters used, etc.) to capture all images in the experiment. Positive and negative controls can be used to optimize the settings. If the number of PLA signals is large, the PLA signals will be fused/bonded together (see Figure 2). This can occur when studying highly expressed proteins, or during overexposed image capture. When setting up image acquisition, careful settings must be made to obtain individual PLA signals. For how to prevent signal fusion and for other possible optimization aspects, see the Duolink® PLA Troubleshooting Guide.

Image Analysis

Now there are several image analysis tools that can be used to quantify PLA signals. The image data can be analyzed to obtain the average fluorescence intensity and/or the total number of PLA signals for each cell or region within the cell (see Figure 3). The reported quantitative data are data in a given experiment relative to technical and/or biological controls. However, reliable quantization is only possible if the PLA signal is not fused and the image pixels are not saturated.

Please refer to the Duolink® PLA Troubleshooting Guide for troubleshooting tips and tips, general troubleshooting instructions, and frequently asked questions.

Please feel free to contact our technical support team or local sales representative for experimental assistance.

Customization Service

Does your experiment require additional customization? Let us do this for you. Learn more about our customized service plans to accelerate your Duolink® PLA project.