Proliferating and apoptotic cells revealed
Labeling of cells and tissues with several distinct fluorescent probes is now a standard lab practice that allows for multiplexing of functional and antibody-based markers. However, there are fields of research and types of analyses that are much more amenable to colorimetric labeling. Colorimetric detection is far more widespread when studying highly autofluorescent tissue, such as heart, because this autofluorescence can significantly interfere with a fluorescent probe signal. Colorimetric detection is also favored when labeling must be compatible with standard histological stains that provide morphological and contextual information. Here we describe the conversion of our popular fluorescence-based click chemistry assays for cell proliferation and apoptosis to colorimetric assays.
Adapting the click reaction for colorimetric detection
The most direct assay for cell proliferation is the measurement of new DNA synthesis via incorporation of a thymidine analog into the growing DNA strand during the S phase of the cell cycle. In the click chemistry–based cell proliferation assay, the thymidine analog EdU (5-ethynyl-2´-deoxyuridine) is incorporated into newly formed DNA . After cells are fixed and permeabilized, the incorporated EdU is detected by a brief click reaction with a small fluorescent azide, resulting in the covalent attachment of the fluorophore to the DNA. To convert this simple and elegant reaction to a colorimetric signal requires the attachment of an enzyme such as horseradish peroxidase (HRP) to the incorporated EdU, which can then enzymatically convert a substrate such as diaminobenzidine (DAB) to a colored product that is deposited locally and visualized with a white-light microscope.
Unique advantages of colorimetric click reactions
With the development of colorimetric click assays, the click chemistry technology is now accessible to labs that have a brightfield microscope; no specialized fluorescence equipment or training is required. In addition, colorimetric signals create a permanent record on the microscope slide that is not susceptible to fading or photobleaching, allowing results to be viewed immediately after staining or at some later date. Furthermore, EdU-labeled cells detected with a colorimetric DAB reaction can be subsequently treated with standard histology stains—for example, hematoxylin and eosin (H&E)—to reveal the context and location of DNA replication with brightfield illumination.
Colorimetric options for both Click-iT EdU proliferation assays and Click-iT TUNEL apoptosis assays
Detection of new DNA synthesis with the Invitrogen™ Click-iT™ EdU Colorimetric IHC Detection Kit is accomplished with two rapid and essentially irreversible reactions. The click reaction between the incorporated EdU and biotin azide forms a covalent triazole ring, which can then bind a streptavidin-HRP conjugate, resulting in the attachment of HRP to the newly synthesized DNA. Incubation of the cells or tissues with a solution of DAB, a widely used chromogenic HRP substrate, results in deposition of the dark brown polymer in the nuclei of proliferating cells (Figure 1A). Moreover, we have shown that the signals from our colorimetric Click-iT EdU assay are highly correlated with those from our fluorescence-based Click-iT EdU assay (Figure 1B). By reacting EdU-labeled tissue with a mixture of biotin azide and an Alexa Fluor™ azide (molar ratio of approximately 2:1), we can detect proliferation on both fluorescence and brightfield imaging platforms using the Invitrogen™ EVOS™ FL Auto two-camera system (which has a monochrome camera for imaging fluorescent signal and a color camera for imaging colorimetric signal) (Figure 1).
The Invitrogen™ Click-iT™ TUNEL Colorimetric IHC Detection Kit, which detects double-stranded DNA breaks that are the hallmark of late-stage apoptosis, uses the same strategy as the Click-iT EdU colorimetric assay. In the case of TUNEL (terminal deoxynucleotidyl transferase (TdT)–dUTP nick end labeling), a TdT-tailing reaction is used to incorporate EdUTP at the 3´ ends of the double-stranded DNA breaks. Once this ethynyl group reacts with biotin azide through a click reaction, labeled DNA is detected after incubation with a streptavidin-HRP conjugate and DAB, resulting in colorimetric detection in the nuclei of late-stage apoptotic cells (Figure 2).
Figure 2. Mouse tissue section labeling with the colorimetric Click-iT TUNEL apoptosis assay. An 8 μm formalin-fixed, paraffin-embedded (FFPE) section of mouse thymus was labeled with DAB using the Click-iT™ TUNEL Colorimetric IHC Detection Kit to reveal apoptotic cells (dark brown nuclei). The tissue was subsequently stained with eosin Y (pink) followed by nuclear counterstaining with methyl green (blue), dehydrated, and hard-mounted in Thermo Scientific™ Cytoseal™ 60 Mounting Medium. The brightfield image was acquired using a 20x objective on the EVOS™ FL Auto Imaging System equipped with a color camera.
Strategies for gaining access to EdU-labeled DNA
The conventional antibody-based BrdU proliferation assay labels newly synthesized DNA with the thymidine analog 5-bromo-2´-deoxyuridine (BrdU) and then detects the incorporated BrdU with a labeled anti-BrdU antibody. Antibody-based cell- and tissue-labeling protocols typically entail some form of antigen retrieval, most commonly heat-induced epitope retrieval (HIER) or protease digestion (PIER), to disrupt fixative-induced crosslinking and restore epitopes so that they can be recognized by the antibody. In addition to HIER, BrdU detection requires that cells or tissues be additionally treated with acid, heat, or DNase to denature the DNA and allow the anti-BrdU antibody access to the incorporated analog.
Because it does not rely on antibodies for detection, fluorescence- based Click-iT EdU detection does not require any extraordinary antigen retrieval or DNA denaturation treatments; for formalin-fixed, paraffin-embedded (FFPE) tissue, deparaffinization and incubation with the small fluorescent azide are all that are needed before imaging the incorporated EdU. For colorimetric Click-iT EdU detection, the pretreatment requirements are intermediate between those for antibody-based BrdU assays and fluorescence-based EdU assays. With colorimetric Click-iT assays, no acid or enzyme treatment is required for DNA denaturation but either HIER or PIER must be applied for the streptavidin-HRP conjugate to gain access to the site of the click-modified DNA. However, the type and duration of this antigen retrieval treatment is very permissive. Although trypsin is supplied in the kit, alternative methods of HIER and PIER have been evaluated and shown to be sufficient to result in detectable EdU signals. For labs that have already determined the HIER methods useful for their antibodies of interest, such protocols will likely also work for colorimetric Click-iT EdU detection. Alternative PIER protocols work equally well—both trypsin or proteinase K digestion have been tested. For delicate tissue like embryonic mouse heart, a short 5-minute protease digestion is sufficient; longer digestion of 20 to 30 minutes is appropriate for more durable tissue like adult rat uterus or intestine.
Contextual information with histology stains
As with any multicolor experiment, detection of multiple targets requires contrasting and non-overlapping colors; when colocalizing two signals, the lighter color is usually assigned to the counterstained background while the darker color is designated for the signal of interest so that it masks the background. The commonly used H&E counterstain works well with DAB-based Click-iT colorimetric assays. Methyl green counterstain also contrasts well with the DAB signal and is compatible with Click-iT EdU and Click-iT TUNEL colorimetric assays, but it requires nonaqueous mounting conditions.
More complex histological stains are useful when identifying key components such as collagen, elastin, muscle, and mucin found in some tissues. The DAB signal produced by the Click-iT EdU colorimetric assay is compatible with Movat’s pentachrome stain—a five-color stain for nuclei and elastin (black), collagen (yellow), ground substance and mucin (blue), fibrin (bright red), and muscle (red)—with only a slight adaptation of the staining protocol. One component of Movat’s pentachrome stain—Verhoeff’s elastin stain—results in black nuclei as well as black elastin. By over-differentiating with the ferric chloride mordant (i.e., washing out the extra black staining of the nuclei by slightly extending the ferric chloride rinse), the nuclei can be lightened to pale gray while the elastin (with its higher affinity for the stain) remains black. This lightening of the nuclear staining allows the dark brown DAB proliferation signal to be seen within the light gray nuclei. Thus, tissues can be click-labeled with EdU or EdUTP for the Click-iT proliferation or apoptosis assay, respectively, and then the labeled DNA is detected using the HRP substrate DAB to produce a dark brown signal, followed by modified Movat’s pentachrome staining, which produces colors that contrast well with the DAB staining. Using this staining strategy, we can detect proliferating cells within the heart valve, in the context of elastin, collagen, and the surrounding muscle tissue (Figure 3A), as well as within intestinal tissue (Figure 3B).
Peyer’s patches exhibit both proliferation and apoptosis
Click chemistry does double duty within special areas of the small intestine containing lymph regions known as Peyer’s patches that function in immune surveillance of and response to the contents of the intestinal lumen. Within the Peyer’s patches, both proliferating cells and apoptotic cells are normally present. Fluorescence-based antibody staining that detects cleaved caspase-3 indicates that there are cells undergoing the early to middle stages of apoptosis, when the caspase family of enzymes is activated (Figure 4A). Colorimetric Click-iT TUNEL labeling also shows the presence of late-stage apoptotic cells that contain fragmented DNA (Figure 4B). Proliferating cells can be detected with colorimetric Click-iT EdU labeling (Figure 4C). When using colorimetric staining for both TUNEL and EdU assays on the same tissue, we recommend first click-labeling the proliferating cells in the Peyer’s patches with EdU and biotin azide and detecting the EdU-labeled cells using streptavidin-HRP and DAB. This EdU assay can then be followed by a second click-labeling reaction of the apoptotic cells by TdT-tailing of the fragmented DNA with EdUTP, followed by reaction with biotin azide and detection with streptavidin- HRP and an HRP substrate that produces a red or purple stain (such as Vector™ NovaRED™ or Vector™ VIP HRP substrates, from Vector Laboratories).
Two-color pulse labeling of DNA with EdU and BrdU
The colorimetric Click-iT EdU assay and the antibody-based BrdU assay can be used together in a dual-pulse labeling experiment, as long as chromatically distinct enzymatic products will be detected. To detect proliferation in tissue that has been pulsed first with EdU and then with BrdU, we used the antigen retrieval and DNA denaturation treatments required for BrdU detection, followed by reaction of EdU with biotin azide and incubation of BrdU with the anti-BrdU primary antibody (typically overnight at 4°C). Next, the tissue was incubated with a mixture of a streptavidin-HRP conjugate (which binds the biotin) and an alkaline phosphatase (AP) conjugate of a secondary antibody (which binds the anti-BrdU primary antibody), followed by their respective substrates, to produce two-color dual-pulse labeling of proliferation (Figure 5). The HRP and AP substrates were chosen carefully to ensure that their products would be chromatically distinct and chemically compatible; note that the AP reaction must be performed in the absence of phosphate buffer. Only a few color combinations permit the determination of colocalization from both chromogens without the use of spectral deconvolution methods .
In an alternative approach, both the EdU and BrdU thymidine analogs can be labeled with HRP, but this labeling must be done sequentially, with an enzyme-quenching step (often with hydrogen peroxide) between successive staining steps. This single-enzyme approach offers a broader choice of colored HRP substrates and better solvent compatibility, but it requires more steps in the detection protocol.
Figure 5. Two-color dual-pulse labeling of proliferating cells in rat tissue using EdU and BrdU. After pulsing with EdU, followed by BrdU 3 days later, rat tissue was pretreated according to a standard BrdU protocol, which requires trypsin digestion followed by HCl denaturation. The Click-iT EdU labeling reaction was performed prior to overnight incubation with an anti-BrdU primary antibody (clone MoBU-1, Cat. No. B35128), which does not cross-react with EdU. Secondary labeling steps containing streptavidin-HRP and alkaline phosphatase (AP) goat anti–mouse IgG antibody were combined. EdU was detected with Vector™ NovaRED™ Peroxidase Substrate (red-brown), an alternative HRP substrate, using the staining protocol for the Click-iT™ EdU Colorimetric IHC Detection Kit, followed by BrdU detection using Vector™ Blue Alkaline Phosphate Substrate (blue). EdU-labeled cells (pulsed first, red-brown) are distributed throughout the vaginal epithelial layer, while BrdU-labeled cells (pulsed 3 days later, blue) appear adjacent to the basement membrane. This stained tissue section was wet-mounted in PBS and imaged using a 20x objective on the EVOS™ FL Auto Imaging System.
Critical input and guidance were kindly provided by the Stankunas and Powell labs at the Institute of Molecular Biology, University of Oregon, Eugene, Oregon.
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