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. 

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

A Navenibody is an antibody conjugated to an oligonucleotide sequence, also known as a “proximity probe”. When two Navenibodies are in close proximity, the oligonucleotides can template circularization of an added pair of oligonucleotides, starting a rolling circle amplification (RCA) reaction leading to a strong and distinct dot. Read more about the technology here

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.

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.

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.

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, Naveni TriFlex Cell MR 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.

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

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

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.

Yes, you can use cells grown on plastic slides

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.

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

Yes, some small changes are needed for optimal performance.

1. Blocking and antibody diluent

Protein target binding specificity may differ depending on the buffers used for blocking and antibody dilution. The NaveniFlex Cell  blocking and antibody/probe diluents , provided in the kit, are compatible with most antibodies. The blocking and diluent composition differs from the original NaveniFlex and the  antibody performance should be re-verified with immunofluorescent staining before proceeding with NaveniFlex Cell experiments.

If the NaveniFlex Cell blocking solution results in significantly reduced signal strength or increased background for a particular antibody, please contact us at contact@navinci.dev.phosdev.se for advice.

2. Antibody concentration

Due to NaveniFlex Cells´ increased sensitivity, the primary antibody concentration should be titrated to determine the optimal dilution for each antibody that gives rise to the highest number of separated signals while keeping the background to a minimum.

Start with the antibody concentration from your previous NaveniFlex experiments.  Perform the antibody titration on both positive and negative controls.

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 or NaveniBright kit:

• MR kits require one mouse and one rabbit antibody
• GM requires one goat and one mouse antibody
• GR requires one rabbit and one goat antibody.

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.

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.

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.

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.

We recommend NaveniFlex Tissue GR for mouse tissue experiment. 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.

Our secondary antibodies are raised in donkey.

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

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

Yes. The Naveni products, with fluorescent readout, have speck-like signals, and each speck can be counted as separate object as long as the proteins of interest are not overly abundant. We recommend quantification by measuring the intensity per cell in each separate channel, or by object segmentation of the speck-like signal and counting. The number of cells can be accounted for by identifying the number of cell nuclei, so do not forget to include a nuclear stain as recommended in the kit instructions.

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.

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.

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.

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.

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

If the NaveniFlex Cell 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.

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

The NaveniFlex Control kit has been designed as a control for the NaveniFlex Cell 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 Cell protocol

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.

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).

With NaveniFlex Cell you can detect protein interactions, analyze endogenous proteins, and study protein modification with the possibility of relative quantification on fixed cells. All NaveniFlex Cell products are based on a secondary antibody system that enables a highly sensitive and specific readout of the selected target(s). You should select which kit to use according to your primary antibody species and preferred filter set for signal detection.

1. According to your antibody species: If you’re working with a mouse and rabbit primary antibody pair, select NaveniFlex Cell MR. Use NaveniFlex Cell GM when working with goat and mouse primary antibodies, and NaveniFlex Cell GR when working with rabbit and goat primary antibodies.
2. According to the filter set on your fluorescent microscope: choose between NaveniFlex Cell RED, with fluorescent dye detectable with the Cy3.5 filter set (Abs max: 595 nm, Exc max: 627), and NaveniFlex Cell Atto647N, with fluorescent dye detectable with the Cy 5 filter set (Abs max: 649 nm; Exc max 662 nm).

There are two ways to co-immunostain with NaveniFlex Cell. Either use a primary antibody directly conjugated to a fluorophore or a primary antibody against your co-stain target together with a secondary antibody conjugated to a fluorophore. If you use the second approach, make sure that the primary antibody targeted by your secondary antibody is not from the same species as the two primary antibodies used for the NaveniFlex Cell assay.

Co-immunostaining should be performed after Reaction 2 (step 6) but before nuclear staining (step 7). Use your internal protocol for the optimal incubation time.

The NaveniFlex Cell kit consists of two boxes shipped separately. Upon receipt, store Box 1 at 4-8 °C and Box 2 at -15 to -25 °C.

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

Yes. The NaveniFlex Cell assay generates dot-like signals, and each dot can be counted as a separate object as long as the proteins of interest are not overly abundant. We recommend quantification by either measuring the intensity per cell in each separate channel or by object segmentation of the dot-like signal and object counting. The number of cells can be accounted for by identifying the number of cell nuclei, so remember to include a nuclear stain as recommended in the kit instructions.

NaveniFlex Cell can be applied to all sample types that can be used for in situ staining with traditional staining methods such as immunofluorescence (IF) and immunohistochemistry (IHC). The samples should be immobilized on slides or cell culture plates, fixed and permeabilized.

No, the NaveniFlex Cell can only be used on fixed cells.

Yes, it is possible, although the NaveniFlex Cell products are optimized and specifically designed for cell-based assays. To perform experiments on tissue, we strongly recommend the NaveniFlex Tissue products. The NaveniFlex Tissue products are designed for tissues, improving sensitivity and removing cell-type specific bbackgrounds seen in many tissue samples.

All NaveniFlex Cell batches are thoroughly quality-controlled and tested with various antibodies on human cell lines to ensure high quality and reproducibility. New batches are always compared against older batches to ensure lot-to-lot consistency.

NaveniFlex Tissue is specifically designed for tissues, where it improves sensitivity and removes high background seen in certain samples. In these problematic tissues, NaveniFlex Tissue is your best choice to obtain the optimal number of signals and image clarity. If you work with cells, NaveniFlex is the kit for you.

The workflow and reagents are optimized to give more sensitive detection in tissue samples. Our proprietary blocking procedures ensure significantly lower backgrounds.

Yes, NaveniFlex Tissue can be used on fresh frozen human and mouse tissues.

Yes, NaveniFlex Tissue can be used on fresh frozen human and mouse tissues. 

Yes, it is possible, although we recommend that you use NaveniFlex for cell samples.

You can choose the NaveniFlex Tissue kit according to your preferred filter set on your fluorescent microscope. Choose between NaveniFlex Tissue RED, with fluorescent dye for the Cy3.5 filter set (Abs max: 595 nm, Exc max: 627), and NaveniFlex Tissue Atto647N, with fluorescent dye for the Cy 5 filter set (Abs max: 649 nm; Exc max 662 nm).

NaveniFlex Tissue is validated on FFPE and fresh frozen human and mouse tissue sections. The product is validated on several types of tissue, both healthy and cancerous e.g., colon, tonsil, ovary, kidney, breast, spleen, brain, skin.

No, only the unspecific background generated by specific cell types within the tissue is blocked. For most tissues, autofluorescence is much lower in intensity than the proximity ligation signals, making them easy to detect. If you have a problem with autofluorescence, we recommend you choose the NaveniFlex Tissue kit with the Atto647N detection fluorophore. If the problem persists, autofluorescence can be identified in the FITC channel and subtracted.

All batches of NaveniFlex Tissue are thoroughly quality controlled and tested with various antibodies on human FFPE tissue samples to ensure high quality and reproducibility. New batches are always compared against older batches to ensure lot-to-lot consistency.

There are two ways to co-stain with NaveniFlex tissue. Either with a primary antibody directly conjugated to a fluorophore or by using a secondary antibody conjugated to a fluorophore. If you use the second approach you must make sure that the primary antibody targeted by your secondary antibody is not from the same species as the two primary antibodies used for the NaveniFlex Tissue assay.

• If you use a primary antibody directly conjugated to a fluorophore for co-staining add the antibody to the detection mix and incubate for 30 minutes. The incubation time can be prolonged if needed.
• If you use a secondary antibody conjugated to a fluorophore add the primary during the post-block step, and the secondary during the detection step. Both the post-block step and detection step can be prolonged if needed.

Follow the provided kit instructions from step 8.4 for the final steps of the protocol.

The Naveni™TriFlex Cell MR reagents have a working volume of 4000µl (sufficient for approx. 100 reactions).

Naveni^TMTriFlex Cell MR is a flexible product and allows you to select your preferred primary antibodies, as long as one of them is raised in mouse, and the other one in rabbit. Everything else is included in the kit.

Unlike our NaveniFlex and NaveniBright product lines, which are exclusively used to study protein interactions or post-translational modifications, Naveni™TriFlex Cell simultaneously detects total protein A, total protein B, and the interaction AB between the two proteins,​ and thereby makes it possible to study the functional states and interplay of proteins.

Naveni™TriFlex Cell is a novel proximity-based technology that can concurrently visualize two proteins in a free and complex state in any cell compartment, thus opening the door to studying their functional states and interplay in response to stimulation/inhibition, cell division, etc.

Just like our other products, Naveni™TriFlex Cell MR detects an interaction whenever the targeted epitopes A and B are no more than 40 nm apart. This proximity signal is visualized by the Cy5 filter set. However, in addition to complexes, Naveni™TriFlex Cell MR detects each protein of interest individually, regardless of whether it partakes in the interaction or not. This allows you to also visualize the total amount of protein A, targeted by your rabbit primary antibody, with the FITC filter set, as well as the total amount of protein B, targeted by your mouse primary antibody, with the Cy3 filter set.

Naveni™TriFlex Cell relies on two user-determined primary antibodies against the targets of interest, and on proprietary TriFlex Navenibodies (antibody-based proximity reagents). Only proteins located at less than 40 nm distance are recorded as interacting. Naveni™ TriFlex detects total protein A, total protein B, and the AB interaction. The detected A, B, and AB signals are amplified and generate fluorescent readout in three channels corresponding to each protein pool. Learn more here

Naveni™TriFlex Cell MR has an unparalleled quick and user-friendly workflow. The total run time depends on your preferred duration of primary antibody incubation, which can vary from 1h at +37˚C up to overnight at +4˚C (recommended). If using the recommended overnight incubation of primary antibodies, the protocol on day one is 1h and on day two the run time is under 4h. If you opt for a 1h at +37˚C primary antibody incubation, the entire assay can be performed in one day with a run time of approximately 6h.

Naveni™TriFlex Cell is a proximity-based kit for the specific and sensitive detection of total protein A and protein B, and their interaction AB.

Yes! However, if your counterstain requires you to use a primary and a fluorescently labeled secondary antibody, the primary antibody needs to be raised in a species different from mouse or rabbit. It can be added after reaction 2. Note that if you are using a fluorophore-conjugated primary antibody, the species of origin does not matter. However, bear in mind that the fluorescent label, be it on a primary or a secondary antibody, needs to have an emission spectrum different from FITC, Cy3, or Cy5, as these are the wavelengths in which Naveni™TriFlex Cell signals A, B and AB will be visible.

No, Naveni™TriFlex Cell cannot be used together with NaveniFlex or similar products.

The kit includes three different dyes, one for each type of signal that Naveni™TriFlex Cell detects. To visualize the rabbit antibody signal, use a filter set with excitation wavelength in the range of 480-490 nm and emission wavelength in the range of 525-535 nm. To visualize the mouse antibody signal, use a filter set with excitation in the range of 545-555 nm and emission wavelength of 575 -585 nm. To visualize the proximity signal, use a filter set excitation wavelength in the range of 635-645 nm and emission wavelength of 665-675 nm.

We suggest that you start by using the lowest recommended concentration for IF by your primary antibody vendor. If further optimization is necessary, the antibodies can conveniently be titrated simultaneously using Naveni™ TriFlex Cell MR until you get a good signal-to-noise ratio.

Check out our webshop for an updated price in your currency.

We at Navinci are dedicated to making products that work well with most microscopes. One of the advantages of Naveni™TriFlex Cell is the fact that it allows you to identify true colocalization independent of microscope resolution. To use the kit, you need a minimum of three filter sets: FITC (excitation 480-490 nm, emission 525-535 nm), Cy3 (excitation 545-55 nm, emission 575 -585 nm), and Cy5 (excitation 635-645 nm, emission 665-675 nm). In addition, using a nuclear stain such as DAPI or Hoechst is recommended.

The signal detected with the FITC filter corresponds to total protein A – the protein target of your rabbit primary antibody. The signal detected with the Cy3 filter corresponds to total protein B – the protein target of your mouse primary antibody. The signal detected with the Cy5 filter corresponds to the interaction between protein A and protein B.

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).

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.

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

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.

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.

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.

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.

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.

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.

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

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.

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.

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

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

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

NaveniLink is a complementary product to the NaveniFlex Cell, NaveniFlex Tissue and NaveniBright kits, which adds extra flexibility by allowing the user to create their own primary or secondary Navenibodies. NaveniLink enables the simple and rapid conjugation of oligonucleotide arms to two antibodies of your choice, independently of host species.

For any new conjugate, initial storage at 4°C is recommended. A preservative may be desirable for long-term storage. Other storage conditions (e.g. frozen at -70°C or at -20°C with 50% glycerol) may also be satisfactory. The best conditions for any new conjugate must be determined by experimentation or by recommendations from the manufacturer. You should also take into account of how the conjugate will be used and avoid adding substances that will need to be removed later, resulting in inevitable loss of conjugate.

• When you want to detect protein-protein interactions/ post-translational modifications and your primary antibodies are from species other than mouse, rabbit, and goat.
• When you want to detect protein-protein interactions/ post-translational modifications and both of your primary antibodies are from the same species.

Yes, it is possible to combine a custom-conjugated antibody with one Navenibody from the NaveniFlex Cell/Tissue or NaveniBright kits, as long as there is no species cross-reactivity.

.

The dilution of your custom-made Navenibody will vary depending on the primary antibody and the application. We recommend that you start with an earlier optimized concentration (via IHC or IF). If you get too low signals or experience a technical background, you may need to do a titration.

If you have custom-conjugated a primary antibody: Add the conjugate(s) in Step 3: Primary antibody incubation and follow the instructions from the kit. We recommend overnight incubation at +4°C. If both primary antibodies are conjugated, skip Step 4: Navenibody Incubation, and proceed directly to Step 5: Reaction 1.

If you have custom-conjugated a secondary antibody: Add the conjugate(s) in Step 4: Navenibody incubation and follow the instructions from the kit.

The antibody concentration should be 1 mg/mL in a suitable buffer (see below). Antibodies that are <1 mg/mL should ideally be concentrated. Buffer pH should be 6.5 – 8.5

Buffer considerations
OK to use with NaveniLink conjugation: Amine-free buffers (e.g. MOPS, MES, HEPES, PBS), non-buffering salts (e.g. sodium chloride), chelating agents (e.g. EDTA), and sugars

Not recommended for NaveniLink conjugation: Thiomersal/ Thimerosal, Merthiolate, BSA, gelatin, tris, glycine, ProClin 300 and nucleophilic components (e.g. amino acids, ethanolamine, mercaptoethanol or DTT)

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.

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).

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.

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.

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.

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

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.

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.

The Naveni PD1/PD-L1 enables you to visualize the interaction between receptor PD1 and its ligand PD-L1 in tissues with intact morphology.

Naveni PD1/PD-L1 can be used with fresh frozen and FFPE tissue material use.

Yes, the kit’s performance is verified in a series of human tissues. See a list and example pictures here

Naveni PD1/PD-L1 is tested but not validated for fresh frozen material use.

No, the Naveni PD1/PD-L1 is not optimized for the detection of the PD1/PD-L1 interaction on mouse tissue.

As a positive control for Naveni PD1/PD-L1, we recommend human tonsil or Hodgkin’s lymphoma tissues.

As a , we recommend a cell system [PDL1 Analyze control cell lines from Histocyte]. PD-L1 analyte control cell lines have a known expression of PD-L1, varying from none to high, and do not express PD1.

One Navenibody binds extracellular PD1 and the other binds the C-terminus on PD-L1.

Naveni PD1/PD-L1 is available with either AP or HRP substrate. If you want to co-stain your assay with directly HRP labeled antibodies select Naveni PD1/PD-L1 AP and if you want to co-stain with directly AP labeled antibodies select Naveni PD1/PD-L1 HRP.

• For Naveni PD1/PD-L1 HRP: quench with hydrogen peroxide (H₂O₂)
• For Naveni PD1/PD-L1 AP: quench with BLOXALL Endogenous Blocking solution

Yes, the Naveni PD1/PD-L1 protocol is already optimized for the best performance. Please see the protocol here

Yes, the following reagents are required but not supplied:

• VectaMount® Express Mounting Medium (H-5700-60) from Vector Laboratories.
• Isopropanol 99,5 %.
• Endogenous peroxidase quenching solution, depending on the kit (AP or HRP).
• TBS and TBS-T – Tris-buffered saline, and Tris-buffered saline supplemented with 0,05% Tween, respectively.

No, we have not observed any need for glycosylation when running Naveni PD1/PD-L1. There is no steric hindrance keeping the antibodies from binding.

No, we do not recommend digital quantification of the Naveni PD1/PD-L1 signals. Make sure to include adequate controls in each experiment to be able to get the most out of your Naveni PD1/PD-L1 results.

Naveni PD1/PD-L1 has a chromogenic read-out, and any type of bright-field light microscope can be used.

We recommend a pH 6 citrate antigen retrieval buffer for Naveni PD1/PD-L1 experiments.

Protocol in short:

• Prepare 1x Citrate Antigen retrieval buffer (Target Retrieval Solution, Citrate pH 6, 10X, C9999, Sigma)
• Boil the slides at 110°C for 10 min and let them rest for approx. 50 min until the temperature is 90-95°C.
• Transfer the hot buffer and sample to a plastic staining jar and let cool for 30 min
• Mix the warm citrate buffer 1:1 with diH₂O in a staining jar, transfer the sample to it, and gradually change to only diH₂
• Wash the slides in staining jars filled with diH₂O five times and then leave the sample in TBS-T for 5 min

Using normal tissue adjacent to the tumor is recommended as biological negative control for the PD1/PD–L1 staining in cancer samples. As a positive control, we recommend human tonsil or Hodgkin’s lymphoma tissue.  

The Naveni PD1/PD-L1 Atto647N enables you to fluorescently visualize the interaction between receptor PD1 and its ligand PD-L1 in tissues while keeping their morphology intact. In contrast to detecting the expression of either protein alone, with this kit you can detect the actual activation of this immune checkpoint axis. 

Naveni PD1/PD-L1 Atto647N can be used both with fresh frozen and FFPE tissue. 

The primary antibodies in the kit are against PD1 (clone EH33, Cell Signaling Technologies) and PD-L1 (clone SP142, Abcam RabMAb®). They were chosen after careful screening and validation and are included at an optimal concentration to ensure robust and reliable results for each staining you perform. 

The PD1 primary antibody binds the extracellular part of the receptor and the PD-L1 antibody binds the C-terminus of the ligand.  

Yes, the Naveni PD1/PD-L1 Atto647N protocol is already optimized for the best performance. Please follow the protocol as described here to ensure that you get reliable data. 

An antibody’s ability to react with the antigen may be lowered by fixation, particularly when a tissue is fixed with formalin, which introduces protein cross-linking. This requires a restoration process called antigen retrieval. Please note that we recommend using a Tris EDTA pH 9 antigen retrieval buffer for Naveni PD1/PD-L1 Atto647N experiments, as we have found it to lead to optimal staining. 

The procedure in short: 

1. Prepare 1X Tris EDTA Antigen retrieval buffer (Antigen retrieval buffer, pH 9.0, 100x, ab93684, Abcam)  
2. Boil the slides at 110°C for 10 min and let them rest for approx. 50 min until the buffer reaches a temperature of about 90-95°C. 
3. Transfer the hot buffer and sample to a plastic staining jar and let cool for 30 min. 
4. Mix the warm antigen retrieval buffer 1:1 with diH2O in a staining jar, transfer the sample to it, and gradually change to only diH2O. 
5. Wash the slides in staining jars filled with diH2O five times and then leave the sample in 1X TBS-T for 5 min. 

Yes, additional IF co-staining can easily be incorporated into the protocol. Just add fluorescently labeled antibodies for co-staining at the detection step (Step8) in the kit instructions. Consider the excitation and emission spectra of the additional fluorophores to avoid spectral bleed-through (we suggest FITC or Cy3). It is not recommended to use fluorescently labeled secondary antibodies for co-staining with the Naveni PD1/PD–L1 Atto647N kit. 

Yes! After imaging, please remove the coverslip, wash off the mounting medium, and start your normal H&E protocol. 

Yes, the kit’s performance is verified in a series of human tissues. See a list and example images here

To visualize Naveni PD1/PD-L1 Atto647N signals, use any epifluorescence microscope with a Cy5 filter set (Abs max: 649 nm; Exc max 662 nm) or a confocal microscope. 

Using normal tissue adjacent to the tumor is recommended as biological negative control for the PD1/PD–L1 staining in cancer samples. As a positive control, we recommend human tonsil or Hodgkin’s lymphoma tissue.  

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

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

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

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

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