Frequently Asked Questions

FAQ Categories

The FAQs are grouped into categories of related topics for your convenience:

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In case your question is not answered here, feel free to contact us directly through e-mail (contact@navinci.se) or by filling in the enquiry form on the support page

What is Proximity Ligation Assay?

Proximity Ligation Assay or Proximity Ligation Technology is a technology for localized protein detection. When pairs of Navenibodies bind protein targets in close proximity, their conjugated oligonucleotides can template circularization of an added pair of oligonucleotides via enzymatic ligation. The circularized DNA strands remain hybridized to the Navenibodies, and one of the oligonucleotides serves as a primer for a localized rolling circle amplification (RCA) reaction. The resulting amplified DNA is detected as a bright fluorescent or chromogenic dot by suitably labeled detection probes. Read more about the technology and the inventors here. 

Which fixation method should I use?

The quality of the staining obtained with the primary antibodies depends on the type of fixative used. The ideal fixative preserves cellular and tissue morphology while rapidly arresting degradative processes such as autolysis, by crosslinking and inhibiting endogenous enzymes. If no particular fixation procedure is recommended for your antibody, then the optimal fixation protocol must be determined for the primary antibody and assay; this may vary depending on the type of cell or tissue as well as your protein of interest. Naveni products is compatible with all fixatives typically used for IHC/IF, including:

  • Ethanol
  • Acetone
  • Zinc
  • Paraformaldehyde (Note: PFA concentration must be determined for each assay)
  • Formalin (10% buffered formaldehyde)

Please refer to the appropriate literature or your primary antibody provider for more specific details.

When different fixation methods are recommended for my antibodies, which one should I choose?

If your antibodies call for different fixation conditions, you may need to prioritize which antibody to use at its optimal requirements. Performing a small-scale test comparing different conditions may be informative before scaling up your experiments.

What antigen retrieval/permeabilization methods can be used with the Naveni products?

Two major antigen retrieval methods are generally used on formalin-fixed paraffin-embedded (FFPE) tissue sections:  Heat-Induced Epitope Retrieval (HIER) and Proteolytic-Induced Epitope Retrieval (PIER). Both methods are compatible with all naveni products. For HIER it may be necessary to optimize the pH of your solution.

Example of buffers and enzymes used for antigen retrieval:

  • HIER: 10 mM citrate buffer, pH 6.0; 1 mM EDTA, pH 8.0 ; Tris-EDTA, pH 9.0; Trilogy™
  • PIER: Proteinase K, pepsin, trypsin

Permeabilization of cellular membranes allows large molecules such as antibodies, oligonucleotides, and enzymes to enter the cell, providing access to intracellular epitopes. It is crucial to optimize the type of permeabilizing agent as well as the exposure time for each experiment.

What controls do I need?

It is important to include experimental controls when running any experiment, including NaveniFlex/Bright, for the results to be properly evaluated. Both negative and positive controls should be included, and these can be either biological or technical.

Biological controls include:

  • Cells and tissues with known expression, or lack of expression, of a particular protein.
  • Systems where the protein expression has either been induced or inhibited (knocked down).
  • Pre-immune serum or a non-specific IgG paired with the antigen-specific antibody (although this is not ideal).

 

Technical controls:

  • For NaveniFlex RM, GM, GR and NaveniBright MR we recommend setting up negative controls by omitting each primary antibody one-by-one. This will provide information on any non-specific binding of the Navenibodies to your primary antibodies.
  • For NaveniFlex MM and RR, the non-primary antibody should be used as a negative control; this will show whether the secondary antibodies (probes) are binding to your tissue/cells.

As a positive control, use two antibodies that recognize two proteins known to interact in the cells or tissues of interest. Alternatively, two antibodies that recognize different epitopes of the same protein may be used, if available.

What are the minimum and maximum distances between two proteins that can be detected by two probes?

The Naveni proximity ligation technology products offers the possibility to visualize the subcellular location of proteins, protein interactions, and protein modifications, provided that the two targeted epitopes are within 40 nm of each other

What reagent volume should I use?

The reaction volume should be adjusted to the sample area. For a reaction area of ca 1 cm2 (typical for grown cells mounted on a chamber slide), a reaction volume of 40 µL is suitable. Ensure that the entire sample is covered with the staining solution.

Reaction volume guidelines

  • 1 cm2 – 40 μl total reaction volume
  • 2 cm2 – 80 μl total reaction volume
  • 3 cm2 – 120 μl total reaction volume
How long can slides be stored before imaging?

If the slides are mounted correctly, they can be stored at +4°C (in the dark) without the risk of losing any of the signal. However, we recommend imaging the slides within a few days of the experiment.

Can I use a plastic slide?

Yes, you can use cells grown on plastic slides

Which species are Naveni products compatible with?

Naveni products are optimized for use in human tissue samples. They may be compatible with tissues from other species, but Navinci Diagnostics cannot guarantee successful staining.

Can I apply Naveni products to my non-fixed / in vivo sample?

No. Naveni technology has been developed and optimized for use in fixed cells and tissues.

Which antibodies are recommended?

To ensure success, antibodies must be carefully selected and parameters including sample fixation, antigen retrieval, blocking, and antibody dilution must be optimized before running a NaveniFlex or NaveniBright assay to ensure optimal antibody binding.

The primary antibodies should be of IgG class, specific for your protein target and preferably affinity purified. They can be either polyclonal or monoclonal antibodies. Always validate the antibody by IF/IHC before carrying out a NaveniFlex or NaveniBright assay.

Tips for choosing antibodies:

  • Ensure that the antibody is suitable for the selected application
  • Find out the immunogen sequence used for antibody production (epitope) – for dual recognition, epitopes must not overlap. If possible, always work with more than one pair of antibodies against your target protein to increase your chance of success.
  • For new antibodies, select antibody test sizes to reduce costs. Many vendors offer this option.

 

Antibody species:

The species of your antibodies should be appropriate for the selected NaveniFlex kit:

  • NaveniFlex MR and NaveniBright MR kits require one mouse and one rabbit antibody
  • NaveniFlex GM requires one goat and one mouse antibody
  • NaveniFlex GR requires one rabbit and one goat antibody.
  • Only one primary antibody is required for the single recognition kits, NaveniFlex MM or NaveniFlex RR (originating from mouse or rabbit, respectively).
Do I need to optimize/titer the primary antibodies?

The primary antibody concentration is an essential determinant for any immunoassay. It should be titrated to determine the optimal dilution for each antibody that gives rise to the highest number of separated signals while keeping background to a minimum.

Start with the antibody concentration from your previous IF or IHC experiments (if applicable), or the concentration recommended in the antibody datasheet. Perform the antibody titration on both positive and negative controls.

How do I detect a protein-protein interaction?

Detection and quantification of interacting proteins can be accomplished using a different primary antibody for each of the two interacting proteins. The antibodies should be from two different species.

How do I detect a protein modification with a dual recognition assay?

Detection and quantification of a protein and its putative posttranslational modification can be done using two different primary antibodies, one directed against the target protein and one against a modification site on the same protein. For phosphorylation studies, for example, you could use a pan-Tyr antibody that recognizes a broad range of tyrosine phosphorylation sites.

How do I detect a single protein with dual recognition?

To detect one protein with the same high level of specificity obtained from a dual recognition assay, two primary antibodies directed against the same protein are required. The two antibodies must be able to recognize their epitopes within the same experimental settings.

How do I detect a single protein with single recognition?

If you have a highly specific primary antibody, single protein recognition can be performed with a single primary antibody against your protein and two navenibodies, both directed against the same host species in which your primary antibody was raised. For single recognition, we offer NaveniFlex RR for rabbit antibodies and NaveniFlex MM for mouse antibodies.

Can I use NaveniFlex MR/GM/MM or NaveniBright MR on mouse tissue?

When mouse antibodies are used on mouse cells or tissue in an immunoassay, this often results in high background signal.

In many cases, background can be reduced by performing an extra blocking step with unconjugated anti-mouse secondary antibodies (preferably Donkey Anti-Mouse) for 1 hour at room temperature. Ensure that the final antibody concentration is not lower than 0.1 mg/mL.  Increasing the blocking time to 2 hours at room temperature or overnight at 4 ºC can be more effective for some assays. In this case, a lower concentration of antibody may be applied. Perform the additional  blocking step after the ordinary blocking step and ensure that the samples are washed before and after.

Which species are the secondary Navenibodies raised in?

Our secondary antibodies are raised in donkey.

Can the two primary antibodies be from the same species?

No, it is not possible to detect protein interactions, modifications, or homodimers using two antibodies from the same species.

Is it possible to do direct conjugation of my antibodies?

It is currently impossible to perform any direct conjugation. Please get in touch with us at contact@navinci.se to discuss potential conjugation services.

Can NaveniFlex/Bright be used to detect multiple protein-protein interactions in one sample simultaneously by incubating the sample with more than two antibodies?

No, this is not currently possible. The kits allow you to detect only one protein-protein interaction per assay.

Can I detect protein-RNA interactions?

It is possible, but there are no quick fixes that will work on all protein-RNA interactions. We recommend using an oligo sequence complementary to the RNA, tagged with a hapten (e.g. biotin), and then adding a mouse/rabbit/goat anti-biotin antibody. You would then be able to detect anti-biotin antibodies in proximity to the protein-specific antibody.

Can I use sheep (ovine) antibodies?

At the moment our kits are only compatible with primary antibodies raised in mouse, rabbit, or goat. However, since sheep and goats are closely related species, the Navenibodies against goat primary antibodies may display some cross-reactivity with sheep antibodies. If your primary antibody works with anti-goat secondary antibodies in an immunofluorescence assay, it should also work with our kit. However, this must be tested for each particular antibody.

Can I use rat antibodies?

At the moment our kits can only be used with primary antibodies raised in mouse, rabbit, or goat. However, since rats and mice are closely related, the Navenibodies against mouse primary antibodies may cross-react with the rat antibodies to some degree. If your primary antibody works with anti-mouse secondary antibodies in an immunofluorescence assay, it should also work with our kit. However, this must be tested for each particular antibody.

Can the protocol be used on whole-mounts or thick tissue sections?

The naveni products are not optimized for use on whole-mount tissues. If you want to stain whole mounts, then the protocol will need to be adjusted. We recommend prolonging each incubation by 50%. An increased enzyme concentration may also be needed.

What is the UnFold technique?

The UnFold technique differs from the traditional PLA technique in that all DNA elements required for amplified detection are included in the oligonucleotides conjugated to the antibodies. The technique is a patented design used as the base for Naveni product development. Read more about the technique here

Can I use a custom blocking/antibody diluent?

Protein target binding specificity may differ depending on the buffers used for blocking and antibody dilution. The NaveniFlex blocking and antibody/probe solutions, provided in the kit, are optimized for NaveniFlex assays and are compatible with most antibodies. However, antibody performance should be verified with immunofluorescent staining before proceeding to NaveniFlex experiments.

If the NaveniFlex blocking solution results in significantly reduced signal strength or increased background for a particular antibody, check the product datasheet for recommendations regarding applicable blocking and antibody diluents.

 

How do I perform co-immunostaining?

A primary antibody derived from a different species than those used in the NaveniFlex kit or an antibody directly conjugated with a fluorophore may be used for co-immunostaining.

Co-immunostaining should be run after Reaction C but before  nuclear staining. The suggested protocol to be applied after Reaction C is as follows:

  • Decant the solution and wash slides for 2 min with 1x TBS in a staining jar under gentle agitation and protected from light.
  • Add and incubate according to your internal protocol
  • Wash slides twice for 2 min each in 1x TBS under gentle agitation
  • Start preparing a nuclear staining solution by diluting DAPI 1:1000 in 1x PBS. Vortex and spin down.
  • Add enough DAPI solution to cover the sample area.
  • Decant the solution and wash slides twice for 2 min each in 1x TBS, followed by a final 15 min wash in 0.1x TBS in a staining jar under gentle agitation.
Which mounting medium can I use?

We recommend Fluoroshield mounting medium (Sigma, F6182). Most mounting mediums compatible with IHC/IF will also work for Naveni products.

What is included in the Control kit?

The NaveniFlex Control kit has been designed as a control for the NaveniFlex MR proximity ligation assay and to help the user distinguish between positive and negative signals. The Control kit contains two primary antibodies against HER2 (raised in mouse and rabbit) and three slides with fixed, pretreated, and dehydrated BT474 cells, and is ready for use with the NaveniFlex MR protocol

What is the difference between the NaveniFlex kits?

With NaveniFlex you can detect protein interactions, analyze endogenous proteins, and study receptor activation with the possibility of relative quantification. All NaveniFlex products are based on the secondary antibody system that enables a highly sensitive and specific readout of the selected target.

NaveniFlex for dual recognition – MR, GR, or GM
The NaveniFlex MR, GR, and GM kits use target recognition by two primary antibodies, enabling the detection of protein interactions, protein modifications, and precise detection of protein expression.

NaveniFlex for single recognition – MM or RR
The NaveniFlex MM and RR kits are used with only one primary antibody, and provide a highly sensitive alternative to traditional IHC for detecting single low abundant proteins. Because the signal is highly amplified, a lower concentration of antibody is required compared to other techniques, meaning that precious antibodies can be used on more samples. NOTE: This protein detection method is only recommended if you have a highly specific primary antibody. If you are uncertain about your antibody specificity, consider using a dual recognition kit with a second primary antibody directed against the same target; this will ensure high assay specificity.

What is the recommended way to report the quantification of Naveni signals?

We recommend using “the number of signals observed per field of view/cell” for reporting Naveni data. If your signals have coalesced and you cannot quantify them separately, try reducing the exposure time during imaging. Imaging software can often identify intensity maxima in an image even though they are too weak to be discerned by the naked eye.
Signal intensity should not be used as a measurable parameter.

How large is a proximity product (amplified signal) and which objective should I use to observe it?

The naveni proximity ligation assay generates distinct, round signals of around 500 nm diameter, which are best visualized using at least a 20X objective (sometimes a 40X may be needed).

Which fluorophores can I select for the experiments and what are their absorption and emission spectra?

The NaveniFlex kit has three different Buffer C vials, each containing a different fluorophore. The fluorophores included in the kit are compatible with the most common filter sets.

The table below can be used to evaluate the compatibility of the fluorophores with the filter settings of your fluorescence microscope. Dye calibration may be necessary for individual fluorescence detection instruments.

Dye properties ATTO 488 Texas Red-X ATTO 647N
Molecular weight
750.2
881
807
Extinction Coefficient
12600
14400
18700
Absorbance Max
502 nm
595 nm
649 nm
Emission Max
522 nm
617 nm
662 nm
Extinction Coefficient
87400
80000
173300
Filter set
FITC 488
Cy3.5/Texas red
Cy5
What is included in the NaveniFlex kit and how long is the kit stable for?

The NaveniFlex kit consists of two boxes, shipped separately: Box 1 is shipped at 4-8 °C and Box 2 is frozen on dry ice. Upon receipt, store Box 1 at 4-8 °C and Box 2 at -15  to -25 °C.

NaveniFlex Box 1 NaveniFlex Box 2
Blocking buffer (1x)
Buffer A (5x)
Primary Antibody Diluent (1x)
Enzyme A (40x)
Probe Diluent (1x)
Buffer B (5x)
Probe 1 (40x)
Enzyme B (40x)
Probe 2 (40x)
Buffer C Atto 488 (5x)
Buffer C TEX615 (5x)
Buffer C Atto 647N (5x)
Enzyme C (40x)

 

When stored as directed, the product is stable for at least three months after receipt. All reagents in Box 2 should be stored at -20 °C; this is especially critical for the enzymes, which should also be kept on ice when removed from the freezer. The probes, antibody and probe diluents, and blocking solutions should be stored at +4 °C.

What is included in the NaveniBright kit?

All reagents needed to perform the assay are included in the kit, except the mounting medium (VectaMount® Express Mounting Medium (H-5700-60) from Vector Laboratories) and quenching agent (Hydrogen peroxide for NaveniBright HRP and Levamisole for NaveniBright AP).

Which quenching agent should I choose?

Tissues and cells may contain endogenous peroxidases or alkaline phosphatase, which can lead to non-specific background staining without proper pretreatment.

Ensure that you are using the appropriate quenching agent for your application

  • For NaveniBright HRP: quench with hydrogen peroxide (H2O2).
  • For NaveniBright AP: quench with levamisole.
  • Dual enzyme blocking solution may be used for either NaveniBright HRP or NaveniBright AP.

Always follow the vendor protocol when you are quenching your slides.  Insufficient quenching could result in non-specific background signal.

Can I use my NaveniBright reagents with NaveniFlex reagents?

No, reagents from the NaveniBright kit should not be mixed or combined with reagents from any other Naveni products.

Can I use my usual blocking and dilution buffers?

We don’t recommended using any buffers or diluents other than those provided in the kit. The NaveniBright blocking and antibody/probe dilution buffers are optimized for use in NaveniBright experiments and are compatible with most antibodies.

Is the kit compatible with other substrates and counterstains?

Yes, NaveniBright kits are designed to be compatible with other substrates. Any substrate compatible with HRP can be used with the NaveniBright HRP kit, and any substrate compatible with AP can be used with the NaveniBright AP kit. Substrate incubation time must be optimized for each assay.

What types of samples has the kit been validated on?

The NaveniBright kit has been validated on

  • Frozen or FFPE fixed cells and tissues
  • Paraformaldehyde fixed cell-lines

It is  highly likely that the kit can be used on any sample types that are compatible with regular IHC.

Is the kit compatible with a fluorescent readout?

Yes, signals developed with AP substrate (NaveniBright AP) will also be visible with a fluorescence microscope. The staining will not be as specific though, as fluorochromes are generally visible over a wide range of fluorescent wavelengths. For nuclear stains or other co-staining, we recommend using fluorophores visible in the Far Red spectra.

Which Mounting Medium should I use?

The VectaMount® Express Mounting Medium (H-5700-60) from Vector Laboratories is required for optimal staining performance. The VectaMount® Express Mounting Medium (H-5700-60) shortens the dehydration and clearing step as well as retaining the staining. Longer dehydration steps in ethanol can reduce staining intensity.

Which kit should I select?

You may take into consideration what type of tissue you are studying when choosing NaveniBright kit

Select NaveniBrightfield AP

  • If you are using tissues that have a high level of endogenous peroxide (e.g. bone marrow).
  • If you want to co-stain your assay with directly HRP labeled antibodies.

Select Navenibrightfield HRP

  • If you want to co-stain your assay with directly AP labeled antibodies.
Can I do co-staining?

Yes, the recommended way to perform a co-staining is by using an HRP-labelled primary antibody together with the NaveniBright AP kit.

How should I image my slides?

To obtain the best quality images, we recommend using a brightfield microscope with at least a 20X objective. Ensure you select the correct focal plane so that the signals are in focus. It is important to keep all microscope settings constant throughout an experiment.

White balancing:
When you are imaging in Brightfield, the light that hits the sample is sometimes “warmer” or “colder”. This can cause objects in the image to appear more orange or blue than they actually are. Not all cameras automatically adjust for this. White balancing enables you to define a region of the image background as white, which calibrates the camera to see the true colors in the image.

After imaging, slides should be stored at room temperature.

What substrate development time should I use for the NaveniBright HRP kit?

The optimal developing time for the substrates included in the NaveniBright kits can vary and should be decided by the investigator of each assay. It is important to keep the developing time the same for all slides in any comparison study to avoid the risk of drawing false conclusions.

Note: Extensive development time will result in unspecific background shade covering the tissue.

What is included in the NaveniBright kits and for how long are they stable for

The NaveniBright kits consists of two boxes and two bags, shipped separately: Box 1, Bag 1.2 and Bag 1.3 are shipped at 4-8 °C and Box 2 is frozen on dry ice. Upon receipt, store Box 1 at 4-8 °C and Box 2 at -15 to -25 °C.

NaveniBright HRP

Box 1 Bag 1.2 Bag 1.3 Bag 2
Blocking buffer
HRP Reagent
HRP diluent
NaveniBright Buffer 1 (5x)
Antibody diluent
HRP Substrate 1
Nuclear Stain
NaveniBright Enzyme 1 (40x)
Probe diluent
HRP Substrate 2
NaveniBright Buffer 2 (5x)
Supplement 1
HRP Substrate 3
NaveniBright Enzyme 2 (40x)
Supplement 2
HRP Substrate 4
Probe M
Probe R

 

NaveniBright AP

Box 1 Bag 1.2 Bag 1.3 Bag 2
Blocking buffer
AP Reagent
AP Diluent
NaveniBright Buffer 1 (5x)
Antibody diluent
AP Substrate Diluent
Nuclear Stain
NaveniBright Enzyme 1 (40x)
Probe diluent
AP Substrate 1
NaveniBright Buffer 2 (5x)
Supplement 1
AP Substrate 2
NaveniBright Enzyme 2 (40x)
Supplement 2
Probe M
Probe R

Naveni PTM

How do the Naveni PTM primary Navenibodies bind?

The two Navenibodies bind two individual cytoplasmic epitopes on the RTK, with one Navenibody targeting the protein close to the C-terminal and the other targeting the phosphorylated tyrosine. Navenibodies in close proximity will give rise to an amplified signal, creating a specific and sensitive detection method for phosphorylated RTK’s.

Should I use Naveni PTM or the flexible kits to study protein phosphorylation?

It depends on which phosphorylated protein you are interested in. The Naveni PTM kits target specific phosphorylated proteins (EGFR, HER2, MET, or PDGFR-beta), and the primary antibodies are included in the kits.

If you are interested in a different phosphorylated protein, we recommend using one of our NaveniFlex kits. NaveniFlex kits allow you to detect phosphorylation of any protein, provided that primary antibodies raised in mouse, rabbit, goat, or a combination of these, are available for your target protein (antibodies not included in kit).

Do I need to know in advance which residue is phosphorylated?

No. The Navenibody which binds to the phosphorylated part of the receptor is a pan tyrosine antibody with a high affinity for  a broad range of phosphorylated tyrosine residues. The dual recognition approach of Naveni PTM, utilizing a generic pan antibody  with a receptor-specific antibody, produces highly specific staining.

Can I use Naveni PTM on mouse cell lines?

We don’t recommend using non-human cell lines or tissues with the Naveni PTM kits. The antibodies included in the kits do not cross-react with species other than human.

What kind of cells can I use with the Naveni PTM kits?

The kits have been developed for use on fixed cells. Any cells that are typically used in other immunostaining methods such as IHC or IF can be used in a Naveni PTM assay.

What do I need to run a PTM assay?

In addition to the Naveni PTM detection kit, you will need fixed and pre-treated cells, nuclear staining reagent (DAPI), and traditional immunostaining equipment.

What is included in the Naveni PTM kit, and what is the product stability time?

Naveni PTM kits consist of two boxes that are shipped separately: Box 1 is shipped at 4-8 °C and Box 2 is shipped on dry ice. Upon receipt, store Box 1 at 4-8 °C and Box 2 at –15 to -25 °C.

Box 1 Box 2
Blocking buffer (1x)
Buffer A (5x)
Probe Diluent (1x)
Enzyme A (40x)
Primary Navenibody 1 (40x)
Buffer B (5x)
Primary Navenibody 2 (40x)
Enzyme B (40x)
Buffer C Atto 488 (5x) (Available on request)
Buffer C TEX615 (5x)
Buffer C (Atto 647N (5x) (Available on request)
Enzyme C (40x)

 

When stored as directed, the product is stable for at least three months after receipt. All reagents in Box 2 should be stored at -20 °C; this is especially critical for the enzymes, which should also be kept on ice when removed from the freezer. The probes, antibody and probe diluents, and blocking solutions should be stored at +4 °C.

Ordering

Where can I find kit instructions?

You can find kit instructions for the individual kits on our product page

How do I place an order?

You can either plance an order by using our webshop or by contacting our sales representatives directly at order@navinci.se

How much do your kits cost?

Visit our webshop to see the product list prices or contact us at order@navinci.se if you wish to obtain a personal quote.

Do you have a distributor in my country?

Please contact us directly at contact@navinci.se or on our webpage at navinci.se/distributors/ to find out if we have a local distributor in your area.

What is your standard delivery time?

Products are typically delivered within one week from ordering within the EU and within ten days from ordering for the rest of the world.