# Nuclear Staining: Principle, Types of Stains, Protocol, and Applications > Nuclear staining labels cell nuclei for microscopy and analysis. Learn its principle, common stains such as DAPI and Hoechst, protocol, uses, and limitations. Canonical URL: https://biologynotesonline.com/nuclear-staining-principle-procedure-uses/ Author: Sourav Pan Last updated: September 17, 2026 ![Nuclear Staining: Principle, Types of Stains, Protocol, and Applications](https://biologynotesonline.com/wp-content/uploads/2025/11/Mechanisms-of-Nuclear-Staining-with-DAPI-Hoechst-PI-and-Hematoxylin.webp) ## What Is Nuclear Staining? Nuclear staining is a staining method used to make the cell nucleus visible by labeling the nuclear material, mainly DNA and chromatin. Fluorescent dyes such as DAPI and Hoechst bind with DNA and give a distinct nuclear signal. In this staining, the labeled part is mainly the nucleus, not the complete cell. If cytoplasm, plasma membrane, or another cellular structure is also required for visualization, a different stain or a specific marker is needed. Nuclei serve as useful cellular “landmarks” in microscopic images. The position of nuclei can be picked out and used for locating and counting the cells. Nuclear detection is frequently used as a starting point during image segmentation, after which another fluorescent or histochemical signal can be related with the position of individual nuclei. ### What nuclear staining reveals The position and number of nuclei can be observed in cells as well as within a tissue section. Individual nuclei can be counted and their spatial arrangement can be followed. Cells having more than one nucleus can also be recognized, when the separate nuclei are clearly resolved by the staining and microscopy method. Nuclear staining also brings out nuclear morphology and chromatin organization. The size and shape of nucleus, lobulation and differences in the chromatin distribution or condensation may appear as changes in the nuclear outline and staining texture. Chromatin organization is closely related with nuclear morphology. These nuclear features vary between different cell types and also under different cellular conditions. DNA-binding stains give DNA-associated signals. DAPI, for example, preferentially binds to A-T rich regions present in the minor groove of double-stranded DNA. Its fluorescence becomes much stronger after binding with DNA. Under suitable staining conditions and quantitative imaging, DNA-binding fluorochromes can also be used for measurement of the nuclear DNA content. ### Nuclear stain vs nuclear counterstain When the nucleus, nuclear morphology or nuclear DNA itself is being examined, a nuclear stain is used as the main label. In such experiments, the nuclear staining is the primary signal being studied. A nuclear counterstain is used in a different way. It is added along with or after another stain, antibody, chromogen or fluorescent marker, mainly to show the location of nuclei and give a reference for the main staining signal. DAPI is commonly used as a nuclear counterstain during immunofluorescence, while hematoxylin can be used as the nuclear counterstain during immunohistochemistry. The same nuclear dye may be used as the primary nuclear label in one experiment. In another experiment, it can be used as a counterstain depending on which signal is being studied. ## Principle of Nuclear Staining ![Nuclear staining mechanisms showing DAPI and Hoechst binding the DNA minor groove, propidium iodide intercalating nucleic acids, and hematein–mordant staining nuclear chromatin.](https://biologynotesonline.com/wp-content/uploads/2025/11/Mechanisms-of-Nuclear-Staining-with-DAPI-Hoechst-PI-and-Hematoxylin-1024x576.webp)Nuclear staining mechanisms showing DAPI and Hoechst binding the DNA minor groove, propidium iodide intercalating nucleic acids, and hematein–mordant staining nuclear chromatin. The principle of nuclear staining is based on selective association of a stain with the nucleus or with its components. After staining, the nucleus appears colored or fluorescent and can be distinguished from the surrounding cytoplasm. Nuclear material contains large amount of nucleic acids, mainly DNA. These molecules provide the binding sites for different nuclear stains. Due to chemical affinity towards these components, the stain becomes concentrated in the nuclear region. All nuclear stains do not follow the same mechanism of binding. Depending on the stain, the interaction may occur by electrostatic attraction, DNA groove binding, intercalation between the base pairs, or by binding through a mordant-dye complex. In conventional histological staining, the nucleus is generally basophilic. Nucleic acids contain negatively charged phosphate groups, and basic or positively acting dyes have affinity towards such acidic cellular components. Hematoxylin is a common example, but its staining action is somewhat different. Oxidized hematoxylin (hematein) is normally used together with a metal mordant. The mordant helps the dye complex to bind with the nuclear material. Nuclear stains, therefore, do not belong simply to one chemical class called “basic stains”. Many of the routine nuclear stains behave as basic stains or basic dye-mordant complexes. Fluorescent nuclear dyes are better described according to their particular DNA-binding properties. Acid dyes generally have greater affinity towards positively charged proteins, including many cytoplasmic proteins. DNA-binding fluorescent dyes form a fluorescent association with the nuclear DNA. DAPI (4′,6-diamidino-2-phenylindole), for example, binds mainly to A-T rich sequences present in the minor groove of DNA and forms its characteristic fluorescent complex. Hoechst 33342 also binds preferentially with A-T rich regions of the DNA minor groove. Its fluorescence increases greatly after binding with DNA. A different type of binding occurs with dyes such as propidium iodide (PI). It binds double-stranded nucleic acids mainly by intercalating between the base pairs. PI can bind both DNA and RNA. When DNA-specific measurement with PI is required, RNase treatment is used. ## Common Nuclear Stains Different stains are used for nuclear staining. The selection of stain depends on the staining method and the type of microscopy. Hematoxylin is commonly used in routine histology, while DAPI and Hoechst dyes are widely used for fluorescent nuclear staining. ![Comparison of common nuclear stain choices showing Hoechst for live cells, DAPI for fixed-cell imaging, hematoxylin for histology, and PI with RNase for DNA-content analysis.](https://biologynotesonline.com/wp-content/uploads/2025/11/Choosing-a-Nuclear-Stain-for-Live-Cells-Fixed-Cells-Histology-and-Flow-Cytometry-1024x576.webp)Comparison of common nuclear stain choices showing Hoechst for live cells, DAPI for fixed-cell imaging, hematoxylin for histology, and PI with RNase for DNA-content analysis. Some of the common nuclear staining dyes are- - Hematoxylin- It is one of the most commonly used nuclear stains in histology. It is widely used in [hematoxylin and eosin (H&E) staining](https://biologynotesonline.com/hematoxylin-and-eosin-he-staining-principle-procedure-result-uses/). Hematoxylin is converted into hematein and generally used together with a metal mordant, such as aluminum. The nuclei appear blue to blue-purple after staining and bluing. - DAPI (4′,6-diamidino-2-phenylindole)- DAPI is a fluorescent stain used for staining nuclear DNA. It binds mainly with A-T rich sequences present in the minor groove of DNA. After binding, it produces strong blue fluorescence. - Hoechst dyes- Hoechst 33258 and Hoechst 33342 are fluorescent DNA-binding nuclear dyes. They also bind preferentially with A-T rich regions of the DNA minor groove and give blue fluorescence. Hoechst 33342 is membrane permeant and can be used for staining nuclei of living cells. - Propidium iodide (PI)- It is a red fluorescent nucleic acid stain. PI binds with DNA mainly by intercalating between the base pairs. It can also bind RNA. Therefore, RNase treatment is used when DNA-specific nuclear measurement with PI is required. - Methyl green- Methyl green is a traditional nuclear stain having affinity towards DNA. It has been used for selective staining of nuclear DNA. Under suitable conditions, DNA-bound methyl green can also show far-red fluorescence. - Nuclear Fast Red- It is mainly used as a nuclear counterstain in histological staining procedures. The nuclei appear red to pink after staining. It is commonly used along with other histochemical stains where a contrasting nuclear stain is required. ## Requirement for Nuclear Staining The requirements for nuclear staining depend on the type of stain, specimen, and microscopy method. Fluorescent nuclear staining and routine histological staining require somewhat different materials. Some of the important requirements are- - Sample or specimen- Cells, tissue sections, cell smears, or isolated nuclei can be used for nuclear staining. The specimen is generally placed on a clean glass slide or coverslip for staining and microscopic observation. - Nuclear stain- A suitable nuclear staining dye is required. DAPI or Hoechst dyes are commonly used for fluorescent nuclear staining, while hematoxylin is widely used for staining nuclei in histological sections. - Fixative- Fixation is required in many nuclear staining procedures to preserve the cells and nuclear structure. Paraformaldehyde, ethanol, or other suitable fixatives can be used depending on the specimen and staining method. It is not required for every nuclear stain. Hoechst 33342, for example, can also be used for staining living cells. - Permeabilizing agent- Some staining methods require permeabilization so that the nuclear stain can reach the DNA. Triton X-100 is commonly used for this purpose. The requirement depends on the stain and whether the cells are fixed or living. - Staining and washing buffer- [Phosphate-buffered saline (PBS)](https://biologynotesonline.com/physiological-buffer-preparation/) is commonly used for dilution of nuclear dyes and washing the specimen. Excess stain is removed by washing before microscopic observation. - Glass slide and coverslip- Stained specimens are mounted on a microscope slide and generally covered with a coverslip. A suitable mounting medium can be added before placing the coverslip. For fluorescence staining, an antifade mounting medium may be used to reduce loss of fluorescence. - Additional staining reagents- These depend on the nuclear stain being used. Hematoxylin staining may require a differentiation step and a bluing solution, while these reagents are not required for DAPI or Hoechst staining. - Microscope- A suitable microscope is required for observation of the stained nuclei. Hematoxylin-stained nuclei can be viewed using a light microscope. Fluorescent dyes such as DAPI and Hoechst require a [fluorescence microscope](https://biologynotesonline.com/fluorescence-microscopy-principle-parts-uses/) with the appropriate filter system. ## Nuclear Staining Protocol The nuclear staining procedure depends on the nuclear dye, type of specimen, and whether living or fixed cells are used. The staining concentration and incubation period are selected according to the particular dye and its validated protocol. These conditions are not same for all the nuclear stains. ![Nuclear staining workflow showing separate live-cell and fixed-cell preparation paths followed by staining, optional washing, and fluorescence or bright-field imaging.](https://biologynotesonline.com/wp-content/uploads/2025/11/General-Nuclear-Staining-Protocol-for-Live-and-Fixed-Cells-1024x725.webp)Nuclear staining workflow showing separate live-cell and fixed-cell preparation paths followed by staining, optional washing, and fluorescence or bright-field imaging. ### Step 1 - Sample preparation - Prepare the cells, cell smear, or tissue section that is to be stained. During the staining procedure, the sample should remain properly attached with the slide, coverslip, or a suitable imaging vessel. - For staining of fixed cells, the sample is first fixed using a fixative suitable for the experiment. Stains such as DAPI are commonly used with fixed samples. - The fixed cells can be permeabilized when easy entry of the stain into the cell and nucleus is required. Permeabilization is not necessary for every nuclear staining method. DAPI can eventually enter the fixed cells without permeabilization, but this step can make the staining faster. - In case of live-cell staining, fixation and permeabilization are avoided. A membrane-permeable nuclear dye is selected for such cells. Hoechst 33342 is commonly used for staining the DNA of living cells. ### Step 2 - Preparation of nuclear stain - The working solution of the selected nuclear stain is prepared before staining. Depending on the particular protocol, the stock dye is diluted in a compatible buffer, staining solution, or culture medium. DAPI and Hoechst are commonly prepared in buffered solutions such as PBS. - Use the recommended working concentration according to the dye and the type of sample. Further optimization may be required because different cells and tissues do not always stain equally. - Fluorescent nuclear dyes and the prepared staining solutions are kept protected from unnecessary light during their preparation and staining. This reduces the loss of fluorescence before imaging. ### Step 3 - Incubation of the sample - Add sufficient amount of the nuclear staining solution so that the sample is properly covered. The complete specimen should remain in contact with the staining solution. - The sample is then incubated for the required period. One fixed incubation time cannot be used for every nuclear dye, tissue, or cell type. Even DAPI staining protocols use different staining periods according to the sample and experimental method. - Both staining time and dye concentration need to be considered together. Excess dye or prolonged staining can increase the background, whereas insufficient staining may give a weak nuclear signal. The suitable staining condition is generally selected around the validated protocol of the particular dye. ### Step 4 - Washing and imaging - After incubation, remove the staining solution when the procedure requires washing. The sample is washed with an appropriate buffer, commonly PBS, to remove the excess unbound nuclear dye. Some staining methods can be directly imaged without a separate washing step, and in such cases the dye-specific procedure is followed. - For fixed samples, a suitable mounting medium can be used when required. A coverslip is placed carefully over the stained specimen before microscopic observation. - Fluorescent nuclear stains are observed using the fluorescence channel or filter suitable for their excitation and emission. DAPI and Hoechst are commonly detected in the blue nuclear channel, with excitation around the ultraviolet region. - When a chromogenic nuclear stain such as hematoxylin is used, the stained nuclei are observed by [bright-field microscopy](https://biologynotesonline.com/bright-field-microscope/) instead of a fluorescence channel. ### Controls and optimization - An unstained control is prepared in a similar way but without adding the nuclear stain. It is used for identifying autofluorescence and other background signals already present in the sample. - An appropriate staining control can also be included where possible. A sample known to give the expected nuclear staining is processed along with the experimental samples. - Excessive background can be reduced by changing the dye concentration, staining period, and washing steps. Over-staining is avoided especially when the individual nuclear boundaries or staining intensity are to be measured. - Samples that are being compared are processed under the same staining conditions. Dye concentration, incubation time, washing procedure, and the imaging settings are kept consistent between these samples. ## Applications of Nuclear Staining Nuclear staining is used in histology, cytology, fluorescence microscopy, and different cell-based studies. Some of the important applications are- - Used to identify nuclei and study their arrangement in tissues. Hematoxylin shows nuclear size, shape, and chromatin pattern in histopathology. - Used for counting cells from microscopic images. DAPI-stained nuclei can be detected as individual nuclear objects in cultured cells and tissue samples. - Used to study nuclear morphology such as size, shape, fragmentation, and chromatin condensation. These changes can be compared between normal and treated cells. - DAPI and other nuclear stains are used for detection of apoptosis by observing chromatin condensation and fragmentation of nuclei under fluorescence microscopy. - Propidium iodide (PI) is used for cell cycle analysis by measuring cellular DNA content with [flow cytometry](https://biologynotesonline.com/flow-cytometry-principle-process-uses/). Differences in DNA content are used to analyze different stages of the cell cycle. - Used as a counterstain in [immunofluorescence](https://biologynotesonline.com/immunofluorescence-test-principle-protocol-types-uses/). DAPI shows the position of nuclei, while another fluorescent marker shows the target protein or cellular structure. - Nuclear signals are used for image segmentation and detection of individual nuclei or cells in fluorescence and digital microscopy images. - PI is used for [cell death and membrane integrity studies](https://biologynotesonline.com/cell-viability-test-using-propidium-iodide-pi/). It enters membrane-damaged cells and stains their nucleic acids, while cells having intact membrane generally exclude the dye. Nuclear morphology can also be examined with PI staining. ## Common Nuclear Staining Problems Different problems can occur during nuclear staining because of staining time, stain concentration, fixation, washing, and imaging conditions. Some of the common nuclear staining problems are- ![Common Nuclear Staining Problems and Microscopic Appearance](https://biologynotesonline.com/wp-content/uploads/2025/11/Common-Nuclear-Staining-Problems-and-Microscopic-Appearance-1024x427.webp)Common Nuclear Staining Problems and Microscopic Appearance - Weak staining- Low stain concentration and short staining time can give pale nuclei or no clear nuclear signal. Poor penetration of dye and unsuitable fixation also cause weak staining. Wrong excitation or emission filter gives weak fluorescence. - Over-staining- The nuclei become very dark or strongly fluorescent when excess stain is used or staining is continued for longer time. In hematoxylin staining, insufficient differentiation can also give over-stained nuclei. - High background- Excess dye and incomplete washing can leave unbound stain in the sample, producing background signal. Some tissues and fixatives also give background fluorescence. - Uneven staining- Some nuclei stain strongly while others remain weak. It can occur due to uneven fixation, poor stain penetration, difference in section thickness, or improper sample preparation. - Poor nuclear morphology- The nuclei may become distorted, shrunken, or damaged after staining. Improper fixation, harsh sample treatment, and strong permeabilization can affect the nuclear structure. - Photobleaching- Fluorescent nuclear dyes gradually lose fluorescence during observation on long exposure to strong excitation light. The sample is kept protected from unnecessary light before and during imaging. - Autofluorescence- Some tissues naturally produce fluorescence which can overlap with the nuclear signal. Aldehyde fixation can further increase autofluorescence in certain samples. - Wrong fluorescence channel- DAPI and Hoechst are generally viewed in the blue nuclear channel. Use of incorrect excitation or emission filter can produce very weak signal or the stained nuclei may not be visible. ## Limitations of Nuclear Staining Some of the limitations of nuclear staining are- - It mainly gives information about the nucleus. Cytoplasm, cell membrane, and specific proteins cannot be studied by nuclear staining alone. - Some nuclear stains are not completely specific for DNA. Propidium iodide (PI) also binds with RNA. - Many nuclear dyes are commonly used with fixed cells. Their use for living cells is limited and suitable membrane-permeable dyes are required. - Fixation can affect the nuclear staining. It may change dye accessibility, staining intensity, and preservation of the nuclear material. - Fluorescent nuclear stains may undergo photobleaching on prolonged exposure to excitation light. - Strong fluorescence excitation can damage living cells, especially during repeated or long-term imaging. - DNA measurement from staining is affected by several factors. Stain concentration, fixation, chromatin structure, and staining conditions can change the fluorescence intensity. - Autofluorescence of the sample and other fluorophores may interfere with the nuclear fluorescence signal. ## Nuclear Staining at a Glance TopicKey pointsDefinitionNuclear staining is used to stain and visualize the nucleus or its nuclear components, mainly DNA and chromatin.PrincipleIt is based on selective association of a stain with nuclear material. Different stains bind by different mechanisms.Nuclear natureNuclei are generally basophilic because nucleic acids contain negatively charged phosphate groups.Common stainsHematoxylin, DAPI, Hoechst dyes, propidium iodide (PI), methyl green, and Nuclear Fast Red.HematoxylinCommon nuclear stain in routine histology and H&E staining. Nuclei appear blue to blue-purple.DAPIFluorescent DNA stain. It binds mainly with A-T rich regions in the minor groove of DNA and gives blue fluorescence.Hoechst dyesFluorescent DNA-binding dyes. Hoechst 33342 can also be used for staining nuclei of living cells.Propidium iodide (PI)Red fluorescent nucleic acid stain. It intercalates between base pairs and can bind both DNA and RNA.Main requirementsSpecimen, nuclear stain, suitable buffer, fixative when required, permeabilizing agent when required, slide, coverslip, and microscope.Basic procedurePrepare the sample → prepare stain → incubate → wash when required → mount → observe under suitable microscope.Live-cell stainingFixation and permeabilization are avoided. A membrane-permeable nuclear dye is required.Fixed-cell stainingFixation is commonly performed. Permeabilization may be used depending on the stain and sample.ApplicationsHistology, cell counting, nuclear morphology, apoptosis studies, cell cycle analysis, immunofluorescence, and image segmentation.Common problemsWeak staining, over-staining, high background, uneven staining, poor nuclear morphology, photobleaching, and autofluorescence.LimitationsMainly shows nuclear information. Some dyes are not completely DNA-specific, and fluorescent staining may be affected by photobleaching, phototoxicity, fixation, and background fluorescence. ## References - Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Looking at the structure of cells in the microscope. In Molecular biology of the cell (4th ed.). Garland Science. [https://www.ncbi.nlm.nih.gov/books/NBK26880/](https://www.ncbi.nlm.nih.gov/books/NBK26880/) - Carriles, R., Schafer, D. N., Sheetz, K. E., Field, J. J., Cisek, R., Barzda, V., Sylvester, A. W., & Squier, J. A. (2009). Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy. Review of Scientific Instruments, 80(8), 081101. [https://doi.org/10.1063/1.3184828](https://doi.org/10.1063/1.3184828) - Chazotte, B. (2011a). Labeling nuclear DNA using DAPI. Cold Spring Harbor Protocols, 2011(1), pdb.prot5556. [https://doi.org/10.1101/pdb.prot5556](https://doi.org/10.1101/pdb.prot5556) - Chazotte, B. (2011b). Labeling nuclear DNA with Hoechst 33342. Cold Spring Harbor Protocols, 2011(1), pdb.prot5557. [https://doi.org/10.1101/pdb.prot5557](https://doi.org/10.1101/pdb.prot5557) - Ciani, C., Pistorio, G., Mearelli, M., & Falcone, C. (2023). Immunofluorescence protocol for localizing protein targets in brain tissue from diverse model and non-model mammals. STAR Protocols, 4(3), 102482. [https://doi.org/10.1016/j.xpro.2023.102482](https://doi.org/10.1016/j.xpro.2023.102482) - Cossarizza, A., Chang, H.-D., Radbruch, A., Abrignani, S., Addo, R., Akdis, M., Andrä, I., Andreata, F., Annunziato, F., Arranz, E., Bacher, P., Bari, S., Barnaba, V., Barros-Martins, J., Baumjohann, D., Beccaria, C. G., Bernardo, D., Boardman, D. A., Borger, J., . . . Yang, J. (2021). Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition). European Journal of Immunology, 51(12), 2708–3145. [https://doi.org/10.1002/eji.202170126](https://doi.org/10.1002/eji.202170126) - Crowley, L. C., Chojnowski, G., & Waterhouse, N. J. (2016). Measuring the DNA content of cells in apoptosis and at different cell-cycle stages by propidium iodide staining and flow cytometry. Cold Spring Harbor Protocols, 2016(10), pdb.prot087247. [https://doi.org/10.1101/pdb.prot087247](https://doi.org/10.1101/pdb.prot087247) - Cummings, B. S., & Schnellmann, R. G. (2004). Measurement of cell death in mammalian cells. Current Protocols in Pharmacology, 25, 12.8.1–12.8.22. [https://doi.org/10.1002/0471141755.ph1208s25](https://doi.org/10.1002/0471141755.ph1208s25) - Darzynkiewicz, Z. (2011). Critical aspects in analysis of cellular DNA content. Current Protocols in Cytometry, 56, 7.2.1–7.2.8. [https://doi.org/10.1002/0471142956.cy0702s56](https://doi.org/10.1002/0471142956.cy0702s56) - Fischer, A. H., Jacobson, K. A., Rose, J., & Zeller, R. (2008). Hematoxylin and eosin staining of tissue and cell sections. CSH Protocols, 2008, pdb.prot4986. [https://doi.org/10.1101/pdb.prot4986](https://doi.org/10.1101/pdb.prot4986) - Gurina, T. S., & Simms, L. (2023). Histology, staining. In StatPearls. StatPearls Publishing. [https://www.ncbi.nlm.nih.gov/books/NBK557663/](https://www.ncbi.nlm.nih.gov/books/NBK557663/) - Icha, J., Weber, M., Waters, J. C., & Norden, C. (2017). Phototoxicity in live fluorescence microscopy, and how to avoid it. BioEssays, 39(8), 1700003. [https://doi.org/10.1002/bies.201700003](https://doi.org/10.1002/bies.201700003) - Jež, M., Bas, T., Veber, M., Košir, A., Dominko, T., Page, R., & Rožman, P. (2013). The hazards of DAPI photoconversion: Effects of dye, mounting media and fixative, and how to minimize the problem. Histochemistry and Cell Biology, 139(1), 195–204. [https://doi.org/10.1007/s00418-012-1039-8](https://doi.org/10.1007/s00418-012-1039-8) - Kapuscinski, J. (1995). DAPI: A DNA-specific fluorescent probe. Biotechnic & Histochemistry, 70(5), 220–233. [https://doi.org/10.3109/10520299509108199](https://doi.org/10.3109/10520299509108199) - Kobayashi, D., Shibata, A., Oike, T., & Nakano, T. (2017). One-step protocol for evaluation of the mode of radiation-induced clonogenic cell death by fluorescence microscopy. Journal of Visualized Experiments, (128), e56338. [https://doi.org/10.3791/56338](https://doi.org/10.3791/56338) - Lee, C. M. (2016). Identification of kinesin-1 cargos using fluorescence microscopy. Journal of Visualized Experiments, (108), e53632. [https://doi.org/10.3791/53632](https://doi.org/10.3791/53632) - Lin, C.-S., Xin, Z.-C., Dai, J., & Lue, T. F. (2013). Commonly used mesenchymal stem cell markers and tracking labels: Limitations and challenges. Histology and Histopathology, 28(9), 1109–1116. [https://doi.org/10.14670/HH-28.1109](https://doi.org/10.14670/HH-28.1109) - Llewellyn, B. D. (2009). Nuclear staining with alum hematoxylin. Biotechnic & Histochemistry, 84(4), 159–177. [https://doi.org/10.1080/10520290903052899](https://doi.org/10.1080/10520290903052899) - Munyenyembe, K., Timmons, C., Weiner, A. K. M., Katz, L. A., & Yan, Y. (2021). DAPI staining and DNA content estimation of nuclei in uncultivable microbial eukaryotes (Arcellinida and Ciliates). European Journal of Protistology, 81, 125840. [https://doi.org/10.1016/j.ejop.2021.125840](https://doi.org/10.1016/j.ejop.2021.125840) - Neely, A. E., & Bao, X. (2019). Nuclei isolation staining (NIS) method for imaging chromatin-associated proteins in difficult cell types. Current Protocols in Cell Biology, 84(1), e94. [https://doi.org/10.1002/cpcb.94](https://doi.org/10.1002/cpcb.94) - Prieto, D., Aparicio, G., Machado, M., & Zolessi, F. R. (2015). Application of the DNA-specific stain methyl green in the fluorescent labeling of embryos. Journal of Visualized Experiments, (99), e52769. [https://doi.org/10.3791/52769](https://doi.org/10.3791/52769) - Roukos, V., Pegoraro, G., Voss, T. C., & Misteli, T. (2015). Cell cycle staging of individual cells by fluorescence microscopy. Nature Protocols, 10(2), 334–348. [https://doi.org/10.1038/nprot.2015.016](https://doi.org/10.1038/nprot.2015.016) - Skinner, B. M., & Johnson, E. E. P. (2017). Nuclear morphologies: Their diversity and functional relevance. Chromosoma, 126(2), 195–212. [https://doi.org/10.1007/s00412-016-0614-5](https://doi.org/10.1007/s00412-016-0614-5) - Sun, Y., Ip, P., & Chakrabartty, A. (2017). Simple elimination of background fluorescence in formalin-fixed human brain tissue for immunofluorescence microscopy. Journal of Visualized Experiments, (127), e56188. [https://doi.org/10.3791/56188](https://doi.org/10.3791/56188) - Tarnowski, B. I., Spinale, F. G., & Nicholson, J. H. (1991). DAPI as a useful stain for nuclear quantitation. Biotechnic & Histochemistry, 66(6), 296–302. [https://doi.org/10.3109/10520299109109990](https://doi.org/10.3109/10520299109109990) - Tarnowski, B. I., Sens, D. A., Nicholson, J. H., Hazen-Martin, D. J., Garvin, A. J., & Sens, M. A. (1993). Automatic quantitation of cell growth and determination of mitotic index using DAPI nuclear staining. Pediatric Pathology, 13(2), 249–265. [https://doi.org/10.3109/15513819309048211](https://doi.org/10.3109/15513819309048211) - Titford, M. (2005). The long history of hematoxylin. Biotechnic & Histochemistry, 80(2), 73–78. [https://doi.org/10.1080/10520290500138372](https://doi.org/10.1080/10520290500138372) - Toné, S., Sugimoto, K., Tanda, K., Suda, T., Uehira, K., Kanouchi, H., Samejima, K., Minatogawa, Y., & Earnshaw, W. C. (2007). Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis. Experimental Cell Research, 313(16), 3635–3644. [https://doi.org/10.1016/j.yexcr.2007.06.018](https://doi.org/10.1016/j.yexcr.2007.06.018) - Voronin, D. V., Kozlova, A. A., Verkhovskii, R. A., Ermakov, A. V., Makarkin, M. A., Inozemtseva, O. A., & Bratashov, D. N. (2020). Detection of rare objects by flow cytometry: Imaging, cell sorting, and deep learning approaches. International Journal of Molecular Sciences, 21(7), 2323. [https://doi.org/10.3390/ijms21072323](https://doi.org/10.3390/ijms21072323) - Xing, F., & Yang, L. (2016). Robust nucleus/cell detection and segmentation in digital pathology and microscopy images: A comprehensive review. IEEE Reviews in Biomedical Engineering, 9, 234–263. [https://doi.org/10.1109/RBME.2016.2515127](https://doi.org/10.1109/RBME.2016.2515127)