See more - Antibodies validated for tissue clearing and 3D-immunofluorescence microscopy

Background

The standard for microscopic tissue analysis in 2D is based on physical sectioning of larger samples into thin sections and antibody staining via immunohistochemistry (IHC). This method is very time-consuming, costly (as tissue sections have to be prepared and analyzed individually), and most importantly does not represent the 3-dimensionality of biological samples. Also, physical sectioning destroys the sample morphology and by analyzing only a small fraction of a large sample important details such as rare cancer cells are frequently missed1

Recent developments in the field of tissue clearing now allow imaging of large samples, such as biopsies, entire rodent or human organs, or even an entire mouse body in 3D. Tissue clearing aims to render the tissue transparent, thereby reducing light scattering within a sample to a minimum. This enables penetration of light through the sample and thus allows imaging of the entire sample at once without sectioning. Meanwhile, more than 40 different tissue clearing protocols have been described2, 3

However, immunostaining of molecules of interest within large samples, such as thick tissue sections (300 µm - 2 mm) or even entire rodent organs, comes with huge challenges and accounts for the biggest time and cost factors during protocol establishment. A significant number of antibodies have to be screened for functionality for the tissue clearing method and sample of interest. Once a suitable candidate is identified, subsequent titration of concentration and further optimizations are necessary. 

Antibodies pre-validated for 3D immunofluorescence microscopy (3D-IF)

Miltenyi Biotec now offers antibodies that have been specifically developed and validated for 3D imaging of large samples. They are designed to work together perfectly with the MACS Clearing Kit, and are quickly ready to use thanks to easy-to-follow protocols. No matter whether samples are imaged in 3D via light sheet microscopy or confocal microscopy – our antibodies are tested for deep penetration into large samples and specific signals with excellent signal-to-background ratios. As these primary antibodies are conjugated to bright and photostable Vio® Dyes for direct immunostaining, you save 50% of your staining time as compared to indirect staining using secondary antibodies. Crystal clear signals and minimal background are generated by making use of our unique and recombinant REAfinity™ Antibodies, which are engineered for antigen-specificity and minimal background signal. For maximum convenience, we support you along the entire 3D imaging workflow: 

Video
3D-immunofluorescence of a human cerebral organoid stained with Ki-67, β-Tubulin 3 and SOX2 antibodies

Learn why 3D-IF validated antibodies are the most advantageous for your 3D imaging experiments:

Antibody validation for 3D-IF
Figure 1: Antibody validation for 3D-Immunofluorescence

1.) Validated and optimized for deep tissue penetration of large samples cleared with our MACS Clearing Kit

One of the biggest challenges when staining thick tissue sections or entire organs is complete penetration / diffusion of antibodies through the entire sample. Often, the use of non-validated antibodies leads to unsatisfactory signals as antibodies tend to bind non-specifically to the outer surface of samples (fig, 1). As a result, most antibodies have to be screened for compatibility and protocols need to be adapted for the sample and clearing method of interest, resulting in long antibody validation processes and huge costs. 

To ease this burden, our 3D-IF validated antibodies are already screened for functionality, optimized for tissue penetration and titrated for a recommended starting dilution for your experiments in combination with our MACS Clearing Kit.

Antibody diffusion through thick samples
Figure 2: Antibody diffusion through thick samples. 

Depending on the antibody, we perform deep tissue labeling of large samples of different sizes that have been cleared using our MACS Clearing Kit, ranging from spheroids/organoids (200 – 1,000 µm in diameter) to entire mouse brain hemispheres (~4,000 µm in width).

IF antibody workflow
Figure 3: Duration of 3D imaging experiments 

2.) 50% shorter staining duration due to fluorochrome-conjugated primary antibodies

The immunostaining step of whole-mount samples is the most time-consuming step of 3D-IF staining protocols. As an example, indirect immunofluorescence staining of a mouse brain hemisphere using secondary antibodies requires approximately 14 days (total sample preparation time = 20 days). In contrast, direct immunofluorescence staining using 3D-IF validated fluorochrome-conjugated antibodies does not require use of secondary antibodies, thereby reducing the  staining duration by 50% (7 days) and accelerating your imaging experiments significantly (fig. 3). For optimized staining and clearing protocols, visit our protocol website.

Signal-to-background ratios of large, cleared samples stained using direct vs indirect immunofluorescence staining
Figure 4: Signal-to-background ratios of large, cleared samples stained using direct vs indirect immunofluorescence staining. 

3.) Optimal signal-to-background ratios with primary antibodies conjugated to bright and photostable Vio Dyes 

Indirect immunostaining using secondary antibodies is commonly used to obtain bright signals. However, secondary antibodies often account for non-specific signals due to binding to non-target sample molecules, thereby causing low signal-to-background ratios. To overcome this challenge, direct immunofluorescence staining using primary antibodies that are conjugated to bright fluorochromes are a great alternative.

Our 3D-IF validated antibodies are therefore conjugated to very bright and photostable Vio Dyes providing excellent signal-to-background ratios and minimal photobleaching. These antibodies allow the acquisition of images with even better signal-to-noise ratio as compared to the use of secondary antibodies (fig. 4).

Our 3D-IF validated antibodies are therefore conjugated to very bright and photostable Vio Dyes providing excellent signal-to-background ratios and minimal photobleaching. These antibodies allow the acquisition of images with better signal-to-noise ratio than can be achieved with the use of secondary antibodies (fig. 4).

Our Vio Dyes used for 3D-IF antibodies show comparable characteristics to commonly used Alexa Fluor dyes but come with increased photostability, making them highly suited for widely used laser/filter combinations.

NameExmaxEmmaxEmission color*Comparable Alexa Fluor
Vio B515488514GreenAlexa Fluor 488
Vio R667645668Near-IR***Alexa Fluor 647
* Typical emission colors as viewed through conventional fluorescence microscopes with appropriate filters. 
*** Human vision is insensitive to light beyond ~650 nm; for this reason, it is not possible to view near-IR fluorescent dyes. 

Use our fluorescence spectra viewer to compare Vio Dyes with your commonly used dyes and find the perfect combination for your specific needs.

REAfinity recombinant antibody model
Figure 5: REAfinity recombinant antibody model

4.) Recombinantly engineered antibodies for reproducible results and minimal background signals

REAfinity Antibodies are recombinant antibodies that provide superior lot-to-lot consistency and purity compared to monoclonal antibodies derived from mouse or rat hybridomas. They have been recombinantly engineered to produce highly specific antibodies that do not show binding to Fc-receptors of immune cells present in tissue samples. Additionally, they all have the same human IgG1 isotype, providing highly consistent fluorochrome conjugation.

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