Selecting the right antibodies and nanobodies for fluorescent imaging

We're first talking about primary antibodies, but before we go into that, just going to have a quick overview of immunofluorescence and immunohistochemistry. So what do we mean when we refer to immuno in these terms? Immuno simply refers to the binding of an antibody to an antigen. Immunohistochemistry stands for antigen detection within tissues and immunocytochemistry stands for antigen detection within the cells.

The basic premise behind immunofluorescence is where an antibody binds to the desired antigen in your sample. After it, a secondary antibody is then added, which binds to the primary antibody and this secondary antibody contains a fluorophore which emits the specific wavelength of light in response to excitation. Immunofluorescence is special because it allows a multiplex interactions, whereby you can image multiple proteins in the same sample in the same slide. And this is then, providing that your primary antibodies are raised in different hosts or are of different IgG subclasses, and so, therefore, can be targeted by the selective appropriate anti-secondary antibody.

So this is the overall protocol for immunofluorescence. We first start with fixation, which can either be done using organic solvents, such as acetone, or chemical crosslinkers, such as PFA or formaldehyde. If they're using chemical crosslinkers and we want to access intracellular proteins, we then have to have a permeabilization step. This is then followed by blocking to minimize nonspecific binding and [inaudible 00:05:27] antibody incubation. Here we can do either indirect or direct immunofluorescence. Indirect is the more common one where you incubate with your primary and then secondary antibody with multiple wash steps in between to ensure you're removing all your unbound antibodies in the system. And if we're doing direct, then that's just a simple one-step incubation of your primary antibody, followed then by mounting with a decent mounting media, and then microscopic analysis.

So Proteintech, we have product-specific protocols for all our antibodies, and these detail the specific optimal blocking conditions and the dilution and incubation that we found to work best for us during our validation. So we have somewhere to start.

Polyclonal vs Monoclonal vs Recombinant

This is just a table, which is an overview of the different types of antibodies available to you. So the most common ones are polyclonals, and these are often raised in rabbits, and polyclonals are raised against the whole antigen and therefore contain multiple different IgG molecules to different epitopes. And as such, they have a high affinity and high epitope accessibility. However, polyclonals have inherited lot-to-lot variability, as you can never get the same mix of IgGs across between lots due to the nature of how the antibody is developed and you can also get some cross-reactivity between similar proteins in your samples and this will therefore result in a decrease in specificity and could cause higher background.

Monoclonals contain IgGs against just one epitope. They are most commonly made in mice and generated from hybridomas as single plasma B cells. They have lower lot-to-lot variability than polyclonals and also have lower background. The advantage of these is that they can have high background if used in mouse models and also have potentially low affinity due to the fact that you're only recognizing one epitope.

Recombinant antibodies are monoclonal and just recognize one epitope like monoclonals. And these are made slightly differently because they are made from in-vitro cloning of the heavy and light chain of an IgG isolated from either an immunized rabbit or llama, for example. You can also get animal-free recombinant antibodies which are made straight from a DNA library. These recombinants are great for multiplex experiments and have really low lot-to-lot variability as there is no genetic drift from hybridomas. You are just getting these from in-vitro cloning. Due to the fact that there is only one epitope being recognized, you can get decreased target availability and epitope accessibility.

So when do we use our polyclonal antibodies? Polyclonal antibodies are used in the majority of IHC experiments and also as cross-link tissues. And as you can see in this diagram on your right, we have our desired protein in blue of which our polyclonal antibodies are recognizing many different epitopes. However, when we cross-link with PFA, some of these epitopes become hidden by the chemical cross-linking, and therefore, you will get reduced epitope accessibility. So with your polyclonal antibody, you are much more likely to recognize your target protein.

Also, because that we can recognize multiple different epitopes, polyclonals are therefore better for low abundant target proteins and then they can amplify the target signal. These are also more likely to react if looking at rare species. And other how some of the negatives have been using polyclonals is that you can get cross-reactivity and high background.

So when do we use monoclonal antibodies? Monoclonals are best for resource-intensive studies, and this is because they are scalable produced through hybridoma production. They are not recommended for mice models due to the fact you can get high background and you also have solid target coverage so the majority of the targets that you'll be looking for will also be available in the monoclonal format for the majority of your vendors. As I said before, however, the negatives of monoclonal is that they can be sensitive to epitope changes so whether that is through any of your experimental parameters, or just through things like PFA fixation, this is the negative of having only one epitope being recognized.

So when would we like to use recombinant antibodies? The recombinant antibodies are best for high-throughput staining and automation, and they're especially good for really long-term products and this is because they are genetically defined and have no clonal drift. Therefore, between the lots you are going to get the same antibody again and again. They also have engineered specificities. The recombinants can be excellent if you're looking, for example, for just one different protein isotype. And they're also scalable through highly reproducible E.coli production.

One of the negatives of recombinant antibodies is that your target coverage could be limited because at the moment, there aren't that many recombinant antibodies sold by vendors. However, they are being touted as the future of antibodies, so over time, there will be more and more recombinant antibodies to different targets failing.

Direct vs Indirect

But now, just a quick note about fluorophores. Your fluorophores can either be directly attached to your primary antibody or to your secondary antibody and when we're multiplexing, we want to ensure that the different fluorophores that we use have the spectral profile with minimal overlap. So as you can see in our three CoraLite colors here, if you use them together, you are not going to get that much bleed through when you're looking at the different channels.

There are also two different types of, in general, two types of fluorophores available to you. So you have the conventional, traditional fluorophores, which are things such as FITC, but you also have your synthetic fluorophores, which are Alexa Fluor or CoraLite. And these synthetic fluorophores are brighter and more photostable and therefore, they are best for things such as confocal or low abundant targets, especially for confocal, if you are exciting a fluorophore over a long period of time. For example, if you're performing a stitching image, you really want to use a CoraLite or an Alexa Fluor over something such as FITC because otherwise, this means they are less likely to bleach and you're going to get decent imaging over time.

So as I mentioned, we can do direct or indirect staining. So what is direct? Direct is where you have your primary antibody, which is directly conjugated to your fluorophore, and therefore, you don't have to add any secondary in this and you're much more likely to get a specific signal. However, you do lose the amplification effect of your indirect staining. So indirect staining is when you have an unlabeled primary antibody and then you have your secondary antibody, which attaches to the FC portion of your antibody, and that will have the fluorophore attached. And because of this, you can get two, three, four secondary antibodies just attaching to one primary and therefore, you get this amplification of signal.

These are the positives and negatives of direct and indirect. So some of the positives of direct is that you have a shorter and simpler protocol and you also have much-improved background with less cross-reactivity so you aren't getting any non-specific binding of the secondary antibody in your sample. With indirect you have amplified signal and greater flexibility so that you can use your primary antibody with multiple different secondaries to get different colors available for your panel. Although, some of the negatives for this is that it is a slightly longer and more complex protocol and you can get higher background.

So at Proteintech, we have recently launched our range of popular monoclonal antibodies directly conjugated to our CoraLite fluorophores. And these are in 488, 594, and 647, which is far red.

And so, when do we use our CoraLite antibodies? So you can use them for multiplex imaging, cell markers, organelle labeling, and highly concentrated proteins. We're just going to have a look at few of these examples. So CoraLite is excellent. So direct immunofluorescence are excellent for things such as cytoskeleton markers, which is cytokeratin 18, and beta actin in the red on the right here.

We can also look at organelles. So lamin B1 is a nuclear envelope marker, or here on the right, you can see this mouse heart has been stained with N-Cadherin, which is an inherence junction. And you can really nicely see the definition of the different organelles outlined here.

We can also look at specific cell markers. So TUBB3 is a tubulin beta 3, which is a major component of microtubules. And we will [inaudible 00:14:19] GFAP, which is again, a common, a really common marker used in neuroscience because it is an astrocyte marker so that it marks [inaudible 00:14:30] and cells like that.

Here, just a quick summary of part one. So which antibody do we want to use and when? So polyclonals are good for low abundance targets or if we have cross-link fixed tissues and also good for preliminary investigation work. Monoclonals are good for resource-intensive or long-term studies because we get minimal lot-to-lot variability and you can scale up your antibody quite quickly. And again, with recombinant, these are excellent for multiplex studies due to the really low background and also long-term studies due to the lack of lot-to-lot variability over time.

And so which techniques we want to use? So, as I said, direct staining is really good for abundance target and multiplexing. If you want to use multiple different rabbit monoclonals in one sample, then you can use them directly conjugated on that and avoid any issues here. We also have indirect staining, which is the more common one, and these are good if you have medium or low abundant targets.

Part 2: Secondary Antibodies

And now, we're just going to briefly talk about the different secondary antibodies available to you. So what's in a name? I will now go through this extra-long and complicated name of an example of secondary antibody and explain what each section means and how it can apply to your experiments. But first, we're going to start with choosing a host. So choosing the host of your secondary antibody, there's not really any significant advantage in one over the other so long as it is against the host of your primary antibody, for example, anti-rabbit. But you want to make sure that your secondary antibody host is not the same species is your primary antibody host. You don't want to have a rabbit, anti-rabbit on your study.

And also one good thing to note is that you should probably use the serum from your host as a blocking reagent. So in this example, we would use normal goat serum and this therefore, really helps to minimize any nonspecific binding of your secondary antibody in your study.

We can also get different types of purification of your secondary antibodies and in general, all secondaries are screened to the reactivity to your IgG of choice. However, some antibodies go one step further and here they say "cross-absorbed" and this means that there is an additional purification step to filter out antibodies that bind to off-target species. These are most typically used in multiplex staining, however, they're not necessarily needed for all examples, and in most cases, non-cross-absorbed, secondary antibodies will be suitable for you.

We can also get different subclasses and these are based on the isotype of your IgG. And so polyclonals are a mixture of IgGs and so if you say, "IgG heavy and light gene," this means that you will target the mixture of the IgGs. Your species of primary also determines your subclass mixture.

So here, for example, you can see in our example, the monoclonal antibody here, this is a mouse IgG one. And this is because mice have four different IgG subclasses, IgG1, 2a, et cetera. And this is really useful to know because say you have two mouse monos that you're interested in using in your experiment, you can then multiplex those different monos by using secondaries that recognize your specific subclass.

And also, be aware that you can get fragment primary antibodies so these are often called Fab or Fab2 fragments and these are sold as specific fragments and they're excellent for tissue penetration. They're often used as well, because you want to minimize non-specific binding of the FC region of your primary antibody to certain FC receptors that are present on certain cell types. And therefore, if you do this, you want to ensure that you have a secondary antibody, which is specific for your Fab fragment.

Thanks, Rebecca. Before I start with the nanobody part, I just wanted to remind our attendees that they can add any questions they have already in the chat field and we will answer them in a live Q and A session afterwards.

Part 3: Nanobodies

So now, let's come to the nanobody part. And I would like to start with the definition of nanobodies because maybe not everybody here is familiar with the nanobody format. So nanobodies are single-domain antibodies that are derived from alpaca and you see on the left-hand side an alpaca. A nice animal. And alpacas and other Camelid animals have not only the typical IgG antibody format, but they also have a special type of antibodies, which only consists of the antibody heavy chain. They are called, of course, heavy chain antibodies.

And you see here in green, the highlighted small part of the heavy chain antibody, and this upper binding part is the nanobody. The nanobody, or it's also often called VHH, is 15 kilodalton in size and you can easily use it as a research reagent when you couple it, for example, to a fluorescent dye. Then, we call it a nano-booster or a nano-label or also nano-secondary. And then you fuse it to a fluorescent protein, then it's called a chromobody. And both of these types of nanobody reagents can be nicely applied for imaging.

Direct: Nano-Booster & Label

So let's start with the nano-boosters or nano-labels and there, our rule of thumb is the smaller, the better. So again, a nano-booster or a nano-label is a nanobody that is coupled to a fluorescent dye, and it basically works very similar to a conjugated primary antibody. So we developed nano-boosters and nano-labels specifically to general targets like fluorescent proteins, like TFP and RFP. And when the nanobody with a dye binds to GFP and RFP that you're already have in your cell and in your essay, then the nanobody can stabilize, enhance, and reactivate the fluorescent signal.

It's especially useful when you want to have minimal epitope label displacement to your targets with your staining reagent. And we offer nanoprobes against GFP, RFP, vimentin, histone and Spot tag. And the available fluorophores are Alexa Fluor and ATTO dyes.

The benefits of the nano-boosters and nano-labels is really the size. So when do you want to use it? When you want to have your label and your dye very close to your actual target. Because of the small size, so they are only 1/10th the size of a conventional antibody. You get your dye close to the target and you have a minimal epitope label displacement, and therefore, a minimal linkage error.

Due to the small size, the nanobodies can also penetrate better into deeper tissues and because they are monovalent, they do not cluster. So you can easily mix nanobodies with primary and secondary antibodies and there's no cluster formation. And they can also be recombinantly expressed so you have also a lifetime supply without lot-to-lot variations.

The nano-booster and nano-label are suitable for fluorescent microscopy. So for AP fluorescence, but also for confocal and super-resolution microscopy like STED and STORM. And what you get from them is a higher image resolution and a more uniform staining. On the right side, you also have an example of when we applied a nano-booster to a GFP protein. So you can see here on the left side, the signal intensity when you use GFP alone. GFP often tends to bleach and with the GFP booster, then we get at least a two-fold increase in the fluorescent signal. And also it's shown on the fluorescent images. So when you image GFP alone, the image is darker due to the bleaching and with the booster you can recover the fluorescent signal. You can reactivate it and you get a very nice bright staining.

So on the next slide here, I would like to show you three examples of nano-booster and nano-label applications. Let's start with the first one. This is a staining of nucleus and mitochondria with the GFP and RFP booster. So both boosters were applied to the sample at the same time and you can see that you get a very bright, clean image in a confocal microscope. In the middle here, we have an example of super-resolution microscopy. With this time, it was STED microscopy and the vimentin was fused to a citrine, so to a fluorescent protein and the TFP booster can detect the citrine and fusion and you get a very nice image with a high resolution.

And the citrine alone would not survive STED imaging. And on the right side, you see a more special, more advanced application of the booster. This is staining of a whole mouse after tissue clearing. So in this case, first, the GFP booster was applied to the sample, and then the tissue was cleared afterwards. And yeah, this is especially useful when you want to image deep tissue slices and deep tissue sections.

Multiplexing with secondaries: Nano-Secondaries

On our next slide here, we now switch to another product group that I already mentioned, the nano-secondaries. So as the name already indicates, these are nanobodies which work like secondary antibodies. The nanobodies are also fused to fluorescent dyes and they bind to primary IgG antibodies from mouse or from rabbit in a subclass specific manner. Nano-secondaries are conjugated to Alexa Fluor dyes and they are also recombinantly manufactured so you have a minimal lot-to-lot variations and a high degree of labeling. And they are also especially useful for microscopy techniques like confocal and super-resolution microscopy and customers value a lot that you can use them for multiplexing approaches.

On the right side, you can see different benefits of the nano-secondary. Let's first start with faster staining because the nano-secondary is monovalent. You can incubate the secondary or the nano-secondary, and the primary antibody with the sample at the same time. So you can do a short pre-mixing step, and then you can apply the primary and the nano-secondary to the sample. You see here, as an example, we try to stain actin with the sequential staining. So first the primary, and then the second, the nano-secondary. We could get a nice image. And with one step staining above, we could get the equally nice image but you save a lot of time because you don't have the second incubation and the washing step.

Nano-secondaries also provide higher resolutions because of their small size. So with a secondary antibody, you get your label very close... With the nano-secondary, you get your label very close to the target and this is especially needed when you do super-resolution microscopy. And on the right side there, the benefit we would like to show is cleaner images. So with the nano-secondary, you get a very clean image because they are subclass specific, so they only bind to the IgG of one specific subclass. They have a low cross-reactivity and a very low background, and this is all needed for multiplexing approaches.

Now, I would like to show you two examples of multiplexing. So let's first start with a sample where we used different mouse and rabbit primary antibodies. In yellow, we used the rabbit antibody against lamin. In green, we used a mouse IgG1 antibody against COX4, which is a mitochondrial marker. And in magenta, we used a mouse IgG2b primary antibody against tubulin. And we used the corresponding three different nano-secondaries, we incubated this sample at the same time with all the three primaries and all the three nano-secondaries and could get a high-resolution image with no background and no bleeding.

And in my second example, I would like to show you, as we call it, a triple mouse staining. So we used here three different primary antibodies from mouse against lamin, against MOT and against vimentin. And we also, for this example here, we incubated the primary and the nano-secondary at the same time and could get a very nice multiplexing image without any crosstalk or bleeding.

Chromobodies for live cell

Now I would like to come to the chromobodies, which are nanobodies for live imaging. For chromobodies, the nanobody is fused to a fluorescent protein-like GFP, for example, or RFP. And to use the chromobody, you basically get DNA constructs or you get a plasmid, you transfect your cell with a plasmid and the cell expresses the chromobody during the typical cell cycle. The benefit of the chromobody is that you can visualize endogenous proteins in real-time and it's non-invasive because you don't need to label your endogenous protein with tech by CRISPR Cas, for example. And you just have your endogenous protein and you stain it with a nanobody in the cell. And there you have no interference of the target protein and its function.

The chromobodies are available for different endogenous proteins. Actin, for example, PCNA, histone, lamin. So we have different housekeeping genes. And on the right side, you see an example, a short movie here, there the lamin chromobody was used. And yeah, so fuse to achieve P protein and you see it nicely, this can observe the cell division and the cell cycle.

So I would like to summarize the nanobody parts. Chromotek offers nanobody tools for imaging. You can use the nano-boosters and nano-labels for super-resolution microscopy. And when it comes to thicker tissues, nano-secondaries can be used for any application that need the secondary antibody, but they're especially beneficial when it comes to super-resolution and microscopy, and multiplexing. And the chromobodies are useful for lifestyle imaging.

And now, I would like to summarize our whole talk, also the part from Rebecca. And we thought about that we'd give you some recommendations based on a typical antibody workflow. So when you start your experiment, you look at your target, and there, target abundance is usually important. So when the target abundance is low, then it's recommended to use a polyclonal antibody, primary antibody. When the target is high, then you can use a monoclonal or a recombinant antibody. And for difficult targets, the recombinant antibodies work also well. And when you have a high expressing target, then think about, if you use a directly conjugated antibodies, sorry, for example, protein techs CoraLite dyes and the CoraLite conjugates.

Then, when you start your project, you also think about the amount of sample that you have or the project duration. So when you have only a small amount of sample and the project duration is short, then you are perfectly fine with the polyclonal antibody. But when you have a long-term project and when you have a lot of samples then, a monoclonal or recombinant antibody are useful. Regarding the application, for standard applications, you can use polyclonals, monoclonals, or recombinant. When you work with typical tags, then the nano-boosters and nano-labels are helpful. And when you want to observe a target life cell, and so in the living cell, then the chromobodies can be suitable.

And about secondary antibody you have the choice when your primary antibody is polyclonal, then use a secondary antibody that detects the IgG heavy and light chain because you have different clones in a primary clones in your sample. The monoclonal and recombinant, they detect subclass specific. They also are subclass specific which means that the secondary needs to detect a certain subclass. When it comes to multiplexing, then cross-absorbed antibodies are useful and nano-secondaries, and the nano-secondaries are also very helpful for super-resolution microscopy.