Duolink® Tips and Tricks

1. Experiment

Sample and Antibodies

Sample fixed on glass slides

Optimized:

  • Pretreatment
  • Blocking
  • Primary antibodies, diluent, titer

Protocol: Your own or take a working one and optimize
E.g. DAKO Education Guide, IHC world, Nordiq, Abcam etc.

Primary Antibodies:

  • Affinity purified polyclonal
  • Monoclonal (avoid ascites)

Duolink In Situ Assay: Equipment Needed

Biological Controls

Best and recommended controls include:

Positive control:

  • Known protein interaction or modification
  • Overexpression, stimulation

Negative controls:

  • Irrelevant protein (e.g. non-interacting, present in different compartment)
  • Sample not expressing target protein or down-regulated/repressed, silencing, mutation etc.

 

Lower HER2 in MDA Cells

 

 

Duolink In Situ Assay: Technical Controls

Technical negative controls:

  • Leave one primary antibody out
  • Information about unspecific binding of primary antibodies
    • Helpful to find an optimal titer
    • Leave both primary antibodies out
  • Information about non specificity of PLA® probes
    • Do not use unspecific sera!

Duolink In Situ Control Kit:

  • Useful to check assay conditions in your lab
  • Positive control of Detection Reagent

Above: positive control; Below: negative control.
Left: Brightfield; Right: Fluorescence.

 

Common Parameters to Consider

Check antibody titer to obtain specific and distinct staining

 

  • Use appropriate blocking agent. Dilute PLA probes in a buffer containing the blocking agent
  • Never let your sample dry out during the Duolink In Situ assay
  • Gently tap-off excess solutions
  • Wash in at least 70 mL of wash buffers
  • Use Wash Buffers A and B where specified

 

Incubation temperature:

 

Wash temperature:

  • Bring wash buffers to room temperature
  • Perform wash at room temperature

 


Amplification:

Amplify for 100 min to avoid coalescent signals


Proper thawing technique:

Thaw reagents but use freeze block for Ligase and Polymerase

 

 

Image Interpretation: Common Autofluorescence Interference

  • Use Wash B and Duolink In Situ Mounting Medium with DAPI
  • Do not overexpose

 

Autofluorescence

  • Inherent to sample
  • Can be observed in green channel
  • Alternative: Use either FarRed or Brightfield Detection Reagents

 

2. Sample Analysis and Storage

Mounting and storage

  • Duolink In Situ Brightfield Mounting Medium: non-aqueous, xylene based
  • Duolink In Situ Mounting Medium with DAPI: aqueous, contains anti-fade and DAPI for Fluorescence application



Notes:
We have tested many other media and PLA signals fade away with many of them.
Add a few drops and press out any excess media.

 

Image Acquisition: Filters

 

Image Acquisition: Parameters

Brightfield Application

  • Good contrast
  • White background / unstained tissue


Fluorescence Application

  • 20x or 40x objective
  • Do not overexpose
    • Signals can coalesce
    • Can give rise to autofluorescence
  • Z-stacks if possible

 

3. Image Analysis and Presenting Data

Quantification

Compare only samples that have been run in parallel

  • Images taken with same acquisition parameters under same session

Use 20x or 40x

  • 63x or 100x can give nice images for publication but not worth quantifying

Quantification is relative, e.g.

  • Positive vs. negative controls
  • Signals in nuclei vs. signals in cytoplasm


Counterstaining

Add counterstaining after Wash buffer B step in the protocol, wash and mount

 

Presenting Data

  • Zoom in and scale up
  • Increase brightness and contrast for presentations and publications

Zoom in on a region of interest.

 

Scale up

Example on how increasing brightness/contrast (numerical values above) can be useful when presenting data.

 

4. Probemaker

Probemaker: antibody requirements

Good quality antibodies

  • Affinity purified polyclonals
  • Non-ascites monoclonals
  • Non-modified (e.g. no biotinylation)
  • Stock 1 mg/mL
  • Stock buffer additive free, ideally PBS. Pretreatment
Buffer exchange
G25, G50
Small molecules: e.g. azide, Tris
Affinity purification
Protein A, G
Macromolecules: e.g. gelatin, BSA

 

Probemaker: assay

Concentration of conjugated antibody

Dilution of conjugated antibody

  • Custom solutions: blocking agents + Assay Reagent
  • Duolink In Situ solutions: PLA probe Diluent

 

Contact for Support

You are very welcome to contact us for further support requests at techserv@sial.com
When contacting us, please, provide the following information:

  • Description of your assay
  • Images of your results
  • Controls (positive/negative, biological/technical) that have been performed (if any)
  • Previous IF/IHC results (if any)