A method for whole-mount imaging of mouse ear lymphatic vessels in combination with immunofluorescent staining

Abstract

The application provides a method for whole imaging of mouse ear lymphatic vessels by combining immunofluorescence staining, which comprises the following steps: after fixing a sample, the sample is soaked in different aqueous transparent sample pretreatment liquids for multiple times and then washed; the sample is subjected to gradient immersion decoloring and degreasing by using an aqueous transparent degreasing reagent; the sample is incubated at room temperature by using a primary antibody, washed, incubated at room temperature by using a secondary antibody, and washed; the sample is subjected to refractive index matching by using an aqueous transparent refractive index matching reagent to make the sample transparent; and finally, Z-axis shooting is performed under a confocal microscope, a two-photon microscope or a light sheet microscope to obtain the whole imaging of the mouse ear lymphatic vessels. The application is mainly suitable for whole imaging of mouse ear lymphatic vessels, and the sample is processed by combining immunofluorescence staining and aqueous transparent reagents, so that the integrity of the sample can be ensured in the process of tissue transparency.

Description

Technical Field

[0001] This invention relates to the field of three-dimensional imaging technology, and in particular to a method for overall imaging of the lymphatic vessels in a mouse ear combined with immunofluorescence staining. Background Technology

[0002] Lymphatic vessels are the channels through which interstitial fluid returns to the circulatory system. Because lymph is almost colorless and transparent, it is invisible to the naked eye. Lymphatic vessels are the main pathway for esophageal cancer metastasis; therefore, observing the morphology of lymphatic vessels may help provide information related to tumor metastasis. Although clinical methods exist such as lymphangiography, SPECT, MRI lymphatic vessel sequences, and local injection of methylene blue or indocyanine green to visualize draining lymphatic vessels, these methods are invasive and have low resolution, making it impossible to observe esophageal mucosal lymphatic vessels with diameters ranging from 10μm to 300μm. Digestive endoscopy is the most direct and clearest technique for observing the esophageal mucosa. Magnifying endoscopy can visualize capillary loops within epithelial papillae of about 10μm, but it still cannot visualize colorless lymphatic vessels.

[0003] Therefore, currently, lymphatic vessels can only be visualized through histopathological methods. Traditional pathological techniques involve preparing paraffin or frozen sections of tissue and then using immunohistochemistry or immunofluorescence to reveal the cross-sections of lymphatic vessels. Later, whole-tissue immunofluorescence imaging techniques based on confocal, two-photon, and light-sheet equipment emerged, allowing observation of the three-dimensional structure of lymphatic vessels in mouse skin or mucous membranes. However, because human esophageal mucosa samples are significantly thicker than mouse tissue, laser penetration is low, and the translucency of esophageal squamous epithelium is also very poor, the above methods cannot visualize lymphatic vessel structures. Therefore, a clearing technique is needed to increase the tissue penetration of the laser.

[0004] Currently, the most mainstream clearing techniques are DISCO and CUBIC, both of which offer good clearing results. DISCO requires tissue dehydration, leading to severe internal tissue deformation and significant fluorescence quenching. The first-generation CUBIC method, on the other hand, results in significant tissue swelling and poor defatting. The modified CUBIC method, however, can maintain normal tissue morphology while achieving good tissue transparency and fluorescence preservation.

[0005] However, the CUBIC method is primarily used in basic animal experiments, and its fluorescent staining mainly relies on transgenic or chemical reagents rather than immunofluorescence staining. Currently, there is no standardized procedure to guide the use of immunofluorescence staining combined with CUBIC clearing treatment of mouse ear samples to visualize the complete lymphatic network morphology. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a method for imaging the overall lymphatic vessels of the mouse ear by combining immunofluorescence staining.

[0007] Therefore, the present invention provides a method for imaging the entire lymphatic vessels of a mouse ear using immunofluorescence staining, the method comprising: (1) Mouse ear samples were dehaired with hair removal cream and washed, and then fixed in 4% paraformaldehyde solution; (2) The fixed sample was soaked and washed in the sample pretreatment solution for decolorization and the sample pretreatment solution for decalcification in turn to obtain the pretreated sample; (3) The pretreated samples were subjected to gradient soaking decolorization and degreasing using degreasing solution A and degreasing solution B; (4) The decolorized and degreased sample is incubated with the primary antibody, washed, and then incubated with the secondary antibody and washed. The primary antibody is an antibody that specifically binds to the target tissue antigen, and the secondary antibody is a fluorescently labeled antibody that can specifically bind to the primary antibody. (5) For samples incubated with primary and secondary antibodies, refractive index matching solution C is used to match the refractive index until the sample is transparent; (6) The obtained completely transparent sample is taken out and stored in imaging solution D. The sample is photographed on the Z-axis under confocal, two-photon or light sheet microscope to achieve overall imaging of the lymphatic vessels of mouse ear.

[0008] In some cases, the sample pretreatment solution for decolorization is prepared by mixing CUBIC-L stock solution and distilled water in a 1:1 ratio. The formula of the CUBIC-L stock solution is urea: 25% (w / w), Quadrol (tetraethylenediamine): 25% (w / w), Triton X-100: 15% (w / w), and the remainder is distilled water.

[0009] In some cases, the sample pretreatment solution for decalcification is a 50% CUBIC-L solution prepared by mixing the aforementioned CUBIC-L stock solution with 0.01M PBS (pH 7.2~7.4) in a 1:1 ratio.

[0010] In some cases, the refractive index matching C solution can be directly used as the stock CUBIC-R+(M) solution, or diluted in small amounts with 0.01M PBS (pH 7.2~7.4) or distilled water as needed. The concentration of the diluted stock CUBIC-R+(M) solution ranges from 98% to 100% (v / v). The CUBIC-R+(M) stock solution formulation is: urea: 25% (w / w), sucrose: 50% (w / w), triethanolamine: 10% (w / w), with the remainder being distilled water.

[0011] In some cases, the samples fixed in step (1) are subjected to enzymatic digestion, i.e., the basement membrane is gently digested by incubation at 4°C with 2.5 U / mL Dispase II for 30 minutes, followed by washing with 0.01M PBS (pH 7.2~7.4) and then epidermal peeling and skin edge excision by micromanipulation.

[0012] In some cases, the target tissue antigen is selected from VEGFR3, PROX1, or a combination thereof.

[0013] In some cases, the fluorescent label is selected from FITC, Cy3, Cy5, Cy7, GFP, phycoerythrin, allophycocyanin, AF488, AF555, AF647, TRITC, or Texas Red.

[0014] The beneficial effects of this invention are as follows: This invention is mainly applicable to the overall imaging of lymphatic vessels in mouse ears. By combining immunofluorescence staining with an aqueous clearing reagent, the sample is processed to ensure its integrity during the tissue clearing process; it increases high resolution and deep penetration, breaking through the limitations of traditional slicing, and realizing the visualization of the complete vascular network of tissues with a thickness of more than 100 μm; it provides precise targeting, that is, by combining specific antibodies (such as PROX1), it effectively eliminates background autofluorescence interference; and it has the potential to standardize drug efficacy evaluation, providing a standardized and visualized efficacy evaluation platform for basic research on vascular diseases and targeted drug screening. Attached Figure Description

[0015] Figure 1 In the first round of experiments, lymphatic endothelial markers were immunofluorescently stained. The marker VEGFR3 showed green fluorescence, PROX1 showed red fluorescence, and after MERGE, it showed yellow fluorescence.

[0016] Figure 2 In the second round of experiments, immunofluorescence staining was performed on lymphatic endothelial markers. The marker VEGFR3 showed green fluorescence, PROX1 showed red fluorescence, and after MERGE, it showed yellow fluorescence.

[0017] Figure 3 In the third round of experiments, immunofluorescence staining was performed on lymphatic endothelial markers, among which the marker VEGFR3 showed green fluorescence.

[0018] Figure 4 The final optimized experimental standardized imaging images are as follows: the left image shows the macroscopic presentation of a mouse ear tissue sample; the right image is a three-dimensional fluorescence imaging image of the overall lymphatic network (PROX1, showing red fluorescence) of a mouse ear taken under a confocal microscope. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0020] Example 1: A method for overall imaging of mouse ear lymphatic vessels using immunofluorescence staining

[0021] The mouse ears were treated with a water-based clearing kit to excite and capture all the fluorescent signals from the lymphatic vessels within the tissue.

[0022] To perform overall imaging of the lymphatic vessels in a mouse ear, this embodiment provides a method for overall imaging of the lymphatic vessels in a mouse ear combined with immunofluorescence staining, including the following steps: 1. Materials taken: Mouse ears were collected after cardiac perfusion. The ear samples were dehaired with hair removal cream, washed to eliminate autofluorescence, and fixed with 4% paraformaldehyde solution. A portion of the tissue was then used for the experiment. The fixed ear tissue was washed with 0.01M PBS (pH 7.2–7.4) for 10 minutes, and this washing was repeated three times.

[0023] 2. Transparency (1) Soak the ear tissue block processed in step 1 in 1.5 ml of water-based clearing reagent sample pretreatment solution (for decolorization) for decolorization, and then wash with 0.01M PBS (pH 7.2~7.4) for 10 minutes. Repeat the washing three times. Use 1 / 2 CUBIC-L (50% v / v) as sample pretreatment solution (for decolorization), which is prepared by mixing CUBIC-L stock solution and distilled water in equal volumes of 1:1.

[0024] CUBIC-L stock solution formulation: Urea: 25% (w / w), Quadrol (tetraethylenediamine): 25% (w / w), Triton X-100: 15% (w / w), balance distilled water. Preparation method: Weigh 25g urea, 24.96g tetraethylenediamine, and 28.8g distilled water into a 100mL blue-capped bottle. Place the bottle on a constant-temperature stirrer and stir at 75℃ and 200rpm to dissolve. After cooling to room temperature, add 15g Triton X-100. After dissolving, place the bottle in a vacuum chamber for degassing at 0.1MPa for 30min. The prepared reagent can be stored at room temperature for one month.

[0025] (2) Remove the ear tissue block and use water-based transparent solution. Immerse the sample in 1.5 ml of the pretreatment solution (for decalcification) to remove calcium, then wash with 0.01 M PBS (pH 7.2-7.4) for 10 minutes, repeating the wash three times. The pretreatment solution (for decalcification) can be a 50% CUBIC-L solution prepared by mixing CUBIC-L stock solution and 0.01 M PBS (pH 7.2-7.4) in a 1:1 ratio. Preparation method: Measure equal volumes of CUBIC-L stock solution and 0.01 M PBS (pH 7.2-7.4), mix thoroughly, and then use.

[0026] (3) Degrease with aqueous clearing reagent A, followed by degrease with aqueous clearing reagent B. Then wash with 0.01M PBS (pH 7.2~7.4) for 10 minutes, and repeat the washing three times.

[0027] Degreasing reagent A (used for degreasing and decolorization): The volume fraction of CUBIC-L is (40-60)%, which can be obtained by diluting the CUBIC-L stock solution with distilled water to a volume fraction of (40-60)%. Degreasing reagent B (for strong degreasing): The volume fraction of CUBIC-L is (90-100)%. It can be obtained by directly using the stock solution of CUBIC-L, or by diluting the stock solution of CUBIC-L with distilled water to a volume fraction of (90-100)%.

[0028] 3. Fluorescent labeling: (1) After the above defatting / washing treatment, the ear tissue block was blocked in the blocking solution for 4 hours, and then washed with 0.01M PBS (pH 7.2~7.4) for 5 minutes. The blocking solution was formulated as a 0.01M PBS (pH 7.2~7.4) solution containing 2%~5% (3% in this example) bovine serum albumin and 0.1%~0.5% (0.3% in this example) Triton X-100. Preparation method of blocking solution: Weigh 3g of BSA and dissolve it in 100mL of 0.01M PBS (pH 7.2~7.4), add 300μL of Triton X-100, and filter sterilize after thorough dissolution or use directly.

[0029] (2) A 647-channel PROX1-labeled antibody was selected, with primary antibody (MA5-35749, Thermo Fisher Scientific) and secondary antibody (A32733, Thermo Fisher Scientific). Primary antibody: Rb-anti-PROX1 (Invitrogen MA5-35749 1:200) Secondary antibody: Cy3-conjugated Goat Anti-Rabbit IgG(H+L) (Proteintech SA00009-21:400) (3) Dilute the primary antibody with antibody diluent. The volume ratio of the primary antibody dilution is 1:100 to 1:500 (1:200 in this example). Completely immerse the ear tissue block that has been blocked and cleaned in step (1) in the primary antibody diluent and incubate it on a shaker at 37°C for 24 hours. Then wash it with 0.01M PBS (pH 7.2 to 7.4) for 10 minutes and wash it 3 times. The antibody diluent is a 0.01M PBS (pH 7.2 to 7.4) solution containing 0.5% to 2% (1% in this example) BSA and 0.05% to 0.2% (0.1% in this example) Triton X-100. Preparation method: Weigh 1g of BSA and dissolve it in 100mL of 0.01M PBS (pH 7.2 to 7.4). Add 100μL of Triton X-100 and use it after it is fully dissolved.

[0030] (4) Dilute the secondary antibody with antibody diluent. The volume ratio of the secondary antibody dilution is 1:200 to 1:800 (1:400 in this example). Completely immerse the ear tissue block that has been incubated with the primary antibody and washed in the secondary antibody diluent. Incubate at 37°C on a shaker for 24 hours. Then wash with 0.01M PBS (pH 7.2 to 7.4) for 10 minutes, and repeat 3 times.

[0031] 4. Refractive index matching (5) After the ear tissue block in step 3 has been incubated with secondary antibody and thoroughly washed with 0.01M PBS (pH 7.2~7.4), add water-based clearing reagent refractive index matching solution C (for refractive index matching) and incubate in a shaker at 37°C in the dark for 2 hours.

[0032] The refractive index matching solution C uses CUBIC-R+(M) stock solution with a concentration range of 98% to 100% (v / v).

[0033] Preparation method: Use CUBIC-R+(M) stock solution directly, or dilute it with a small amount of 0.01M PBS (pH 7.2~7.4) or distilled water as needed.

[0034] CUBIC-R+(M) stock solution formula: urea: 25% (w / w), sucrose: 50% (w / w), triethanolamine: 10% (w / w), balance: distilled water.

[0035] Preparation method: Weigh 25g of urea, 50g of sucrose, and 15g of distilled water into a 100mL blue-capped bottle. Place the bottle on a constant-temperature stirrer and stir at 75℃ and 200rpm to dissolve. After cooling to room temperature, add 10g of triethanolamine. After dissolving, place the bottle in a vacuum chamber to degas at 0.1MPa for 30min. The prepared reagent can be stored at room temperature for two weeks.

[0036] 5. Imaging observation The sample was imaged with water-based clearing reagent D solution (used for sample preservation and imaging), sealed in a confocal imaging dish, and observed using a confocal microscope, two-photon microscope, or light sheet microscope.

[0037] The imaging solution D can be a mounting solution made by mixing Mounting Solution 1 and Mounting Solution 2 in a 7:3 ratio using the second-generation CUBIC-L, R+(M) Trial Kit (FUJIFILM Wako).

[0038] Preparation method: Measure 7 volumes of Mounting Solution 1 and 3 volumes of Mounting Solution 2, mix them thoroughly and then use.

[0039] Example 2 Experiment Optimization

[0040] Mouse ear samples are particularly thin, approximately 100 micrometers. After fixation, the ears were dehaired. To investigate the influence of ear skin tissue on the clearing and antibody staining experiments, enzymatic digestion was employed: the basement membrane was gently digested by incubation with 2.5 U / mL Dispase II at 4°C for 30 minutes. After washing with 0.01M PBS (pH 7.2-7.4), the epidermis was microscopically dissected and the skin edges were excised. Subsequently, the aqueous clearing reagent from Example 1 was used, significantly increasing the contact surface area between the clearing reagent and the antibody, thereby eliminating various interferences from the skin (hereinafter referred to as the exclusion scheme). This exclusion scheme does not rely solely on reagent formulation but establishes a combined pretreatment process of "enzymatic digestion + minimally invasive physical intervention." As a control, this example also established a comparative scheme: mouse ears underwent only routine hair removal and fixation. Due to the physical barrier of the epidermal stratum corneum and dense connective tissue, large molecular weight antibodies had difficulty penetrating deep into the tissue.

[0041] This invention reveals that, in the exclusion scheme, the aforementioned pretreatment and the addition of an aqueous clearing agent effectively break down the physical barrier to antibody penetration. Experimental results show that the antibody can uniformly penetrate to the center of tissue up to approximately 100 micrometers thick, significantly enhancing the continuity and fluorescence intensity of lymphatic vessel structures (such as VEGF / LYVE1 channels), achieving uniform imaging across the entire field of view. In the comparison scheme, experimental results show that the fluorescence signal is limited to the tissue edge, and the lymphatic / vascular network in the central region exhibits false negatives (unstained or extremely weak), failing to achieve true "whole-body imaging."

[0042] Ear tissue contains cartilage, which significantly affects antibody labeling. Immunofluorescence labeling of ear samples often fails to produce complete fluorescent signals. After multiple rounds of cartilage removal experiments, the success rate exhibited a degree of randomness. This randomness arises because the presence of cartilage support within the mouse ear severely hinders deep antibody penetration and laser penetration. In initial exploratory experiments, the inventors attempted physical cartilage removal to allow antibody entry. However, due to the extremely tight bond between the subcutaneous tissue and cartilage in the C57 mouse ear, the physical removal process inevitably involved mechanical stretching, severely tearing and damaging the capillary lymphatic network attached to the cartilage surface. This indicates that the success rate of obtaining complete fluorescent signals using physical removal methods is highly unstable and exhibits significant randomness.

[0043] This invention abandons the high-risk physical removal approach. Through standardized processing of an aqueous clearing reagent system, the reagent can fully penetrate and alter the refractive index of cartilage tissue, achieving optical transparency and opening antibody penetration channels. This method completely eliminates the step of physical cartilage removal, fundamentally eliminating the damage to the in-situ lymphatic and vascular three-dimensional network caused by manual mechanical dissection, and preserving the anatomical integrity of the sample to the greatest extent. After the improvement, the deep labeling of the fluorescence signal achieves uniform and stable imaging, greatly improving the success rate and technical reproducibility of the overall imaging experiment.

[0044] To overcome the interference of the epidermis and cartilage on the experiment, the process conditions of this invention have undergone multiple rounds of optimization and improvement. The experimental process, imaging results, and analysis of each round are as follows: (1) First round of experiments: physical cutting to remove bone In the first round of experiments, to address the issue of the cartilage layer hindering antibody entry, a physical cutting method was used for pretreatment, directly attempting bone removal, followed by routine fluorescent staining. The experimental results are as follows: Figure 1 As shown.

[0045] from Figure 1 The first round of imaging results revealed that after bone removal using the cutting method, the anatomical integrity of the sample was severely damaged due to mechanical stretching and peeling, resulting in an extremely unstable vascular network structure. The images showed very low levels of specific fluorescent marker expression, and the few signals that were present exhibited severe breaks and localized absences. This indicates that relying solely on physical cutting to remove bone directly destroys the in-situ micronetwork structure, not only failing to obtain complete signals but also resulting in extremely unstable experimental success rates.

[0046] (2) Second round of experiments: whole material collection combined with epidermal enzyme digestion Given the tissue damage caused by the first round of bone removal, the second round of experiments adopted a different approach, learning from past mistakes. While preserving intact tissue and cartilage, enzymatic digestion was introduced to gently process the epidermis. The experimental results are as follows: Figure 2 As shown.

[0047] Depend on Figure 2 The experimental results showed that after intact tissue sampling and enzymatic digestion of the epidermis, the integrity of the tissue structure was effectively preserved. In the superficial and peripheral areas, the expression levels of green and red fluorescent markers were significantly improved compared to the first round, and the direction of blood vessels could be basically seen. However, because the cartilage layer in the middle was not specifically treated in this round, the undecalcified cartilage still blocked the antibody from penetrating into the deeper layers and the laser from penetrating, resulting in uneven overall fluorescence distribution and weaker signals in the deeper layers.

[0048] (3) Third round of experiments: introducing chemical decalcification pretreatment and combining it with the selected antibody (preliminary experiment attempt) Building upon the foundation laid in the second round of experiments, the third round focused on adding a decalcification step (immersion in CUBIC-L pretreatment solution for decalcification). Simultaneously, to verify the antibody permeability of deep tissues after decalcification, a preliminary experiment was conducted using a selected endothelial lumen marker antibody. The experimental results are as follows: Figure 3 As shown.

[0049] Depend on Figure 3 As can be seen from the high-magnification local section imaging in this round, the addition of the chemical decalcification step and the combination with the selected antibody significantly overcame the barrier effect of the cartilage layer on the penetration of macromolecular antibodies into the deeper layers. The background autofluorescence in the image was greatly reduced, the signal-to-noise ratio was significantly improved, and the tubular structure of the lymphatic endothelial cell membrane delineated by VEGFR3 was clearer and more continuous, with the microtubule orientation being more completely presented.

[0050] (4) Final optimization experiment: The final standardized procedure, building upon the solid foundation of the successful third round of experiments, involves further optimization of parameters such as reagent incubation time, refractive index matching, and slide sealing, as shown in Example 1, thereby achieving standardized imaging. Experimental results are as follows... Figure 4 As shown.

[0051] Depend on Figure 4 It can be seen that the experimental results optimized based on the third round reached the ideal standardized state. Without any slicing and without completely preserving the 100-micrometer thickness and internal cartilage, the entire lymphatic network was displayed brightly, continuously, and with a three-dimensional effect. This final solution effectively solved the problems of large damage from physical bone removal and shallow fluorescence penetration, achieving high signal-to-noise ratio overall imaging across the entire field of view.

Claims

1. A method for imaging the entire lymphatic vessels of a mouse ear using immunofluorescence staining, characterized in that, The method includes: (1) Mouse ear samples were dehaired with hair removal cream and washed, and then fixed in 4% paraformaldehyde solution; (2) The fixed sample was soaked and washed in the sample pretreatment solution for decolorization and the sample pretreatment solution for decalcification in turn to obtain the pretreated sample; (3) The pretreated samples were subjected to gradient soaking decolorization and degreasing using degreasing solution A and degreasing solution B; (4) The decolorized and degreased sample is incubated with the primary antibody, washed, and then incubated with the secondary antibody and washed. The primary antibody is an antibody that specifically binds to the target tissue antigen, and the secondary antibody is a fluorescently labeled antibody that can specifically bind to the primary antibody. (5) For samples incubated with primary and secondary antibodies, refractive index matching solution C is used to match the refractive index until the sample is transparent; (6) The obtained completely transparent sample is taken out and stored in imaging solution D. The sample is photographed on the Z-axis under confocal, two-photon or light sheet microscope to achieve overall imaging of the lymphatic vessels of mouse ear.

2. The method according to claim 1, characterized in that, The sample pretreatment solution for decolorization is prepared by mixing CUBIC-L stock solution and distilled water in a 1:1 ratio. The formula of CUBIC-L stock solution is urea: 25% (w / w), Quadrol (tetraethylenediamine): 25% (w / w), Triton X-100: 15% (w / w), and the remainder is distilled water.

3. The method according to claim 2, characterized in that, The sample pretreatment solution for decalcification was a 50% CUBIC-L solution prepared by mixing the aforementioned CUBIC-L stock solution with 0.01M PBS (pH 7.2~7.4) in a 1:1 ratio.

4. The method according to claim 1, characterized in that, The refractive index matching C solution can be directly diluted with CUBIC-R+(M) stock solution, or diluted in small amounts with 0.01M PBS (pH 7.2~7.4) or distilled water as needed. The concentration range of the diluted CUBIC-R+(M) stock solution is 98%~100% (v / v). The CUBIC-R+(M) stock solution formulation is: urea: 25% (w / w), sucrose: 50% (w / w), triethanolamine: 10% (w / w), with the remainder being distilled water.

5. The method according to claim 1, characterized in that, The fixed sample in step (1) was subjected to enzymatic digestion, that is, the basement membrane was gently digested by incubation at 4°C with 2.5 U / mL Dispase II for 30 minutes, and then washed with 0.01M PBS (pH 7.2~7.4) and the epidermis was peeled and the skin edge was excised by micromanipulation.

6. The method according to claim 1, characterized in that, The target tissue antigen is selected from VEGFR3, PROX1, or a combination thereof.

7. The method according to claim 1, characterized in that, The fluorescent label is selected from FITC, Cy3, Cy5, Cy7, GFP, phycoerythrin, allophycocyanin, AF488, AF555, AF647, TRITC or Texas Red.

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