Labelling

DAPI (hydrochloride) is a blue-emitting fluorescent dye that strongly binds to adenine-thymine-rich regions in DNA. Widely used in fluorescence microscopy and flow cytometry, DAPI serves as a nuclear counterstain. When bound to double-stranded DNA, DAPI exhibits ~20-fold fluorescence enhancement, with an absorption peak at 358 nm and emission peak at 461 nm. It also binds to RNA, albeit with less intensity, causing its emission spectrum to shift to ~500 nm.

Hoechst 33258 is a blue-emitting fluorescent dye that selectively binds to adenine-thymine-rich regions in double-stranded DNA. Its fluorescence is enhanced by AT-rich DNA strands. With excitation/emission maxima at 351/463 nm, it exhibits a considerable Stokes shift. Although it can enter living cells, its permeability is lower than Hoechst 33342. Notably, Hoechst 33258 is less toxic than DAPI, ensuring better cell viability.

Hoechst 33342 is a blue-emitting fluorescent dye which selectively binds to adenine-thymine-rich regions in double-stranded DNA. With excitation/emission maxima at 351/461 nm, it exhibits a considerable Stokes shift. Notably, Hoechst 33342 is less toxic than DAPI, ensuring better cell viability. It is widely used in fluorescence microscopy and flow cytometry for staining chromosomes and nuclei in live and fixed cells, particularly to distinguish condensed pycnotic nuclei in apoptotic cells and for cell sorting.

Hoechst 34580 serves as a blue-emitting fluorescent dye that selectively binds to adenine-thymine-rich regions in double-stranded DNA. It exhibits excitation/emission peaks at 380/438 nm when bound to DNA, with fluorescence intensity sensitive to solvent pH. Unbound dye emits green light in the 510–540 nm range, more pronounced at higher concentrations or inadequate washing. While capable of penetrating living cells, it is less permeable than Hoechst 33342. Importantly, the dye is less toxic than DAPI, ensuring better cell viability post-staining.

Hoechst S769121 (Nuclear Yellow) is a cell-permeable yellow-emitting fluorescent dye that strongly attaches to adenine-thymine-rich regions within double-stranded DNA's minor groove. This dye finds application in fluorescence microscopy, fluorometry, and flow cytometry for staining and measuring DNA content in live and fixed cells. It is frequently paired with retrograde tracers like True Blue for dual-color neuronal imaging. Additionally, Nuclear Yellow can photoconvert diaminobenzidine (DAB) into an electron-dense reaction product, extending its utility to light and electron microscopy.

Propidium iodide (PI) is a red-fluorescent nuclear and chromosome counterstain. It is not permeant to live cells, making it useful for detecting dead cells. PI binds to DNA by intercalating between the bases, with little or no sequence preference. In an aqueous solution, PI has a fluorescent excitation maximum at 493 nm (blue-green) and an emission maximum at 636 nm (red). It is commonly used in flow cytometry for cell viability and DNA content analysis, as well as in microscopy to visualize the nucleus and other DNA-containing organelles.

4-Di-16-ASP serves as a dialkylaminostyryl dye that fluoresces when inserted into membranes or diluted into organic solvents. Specifically, it uniformly labels neurons through diffusion in the plasma membrane. Notably, in intact tissue, the dye does not transfer from labeled to unlabeled cells, although some transfer may occur when the membrane is disrupted (e.g., after sectioning). Its excitation maximum is 492 nm, and its emission maximum is 607 nm.

Cy5 DIC18 is a far-red fluorescent carbocyanine dye used for cell membrane labeling both in vivo and in vitro. It diffuses laterally to stain the entire cell, making it suitable as an anterograde and retrograde neuronal tracer. Notably, in intact tissue, the dye does not transfer from labeled to unlabeled cells, although some transfer may occur when the membrane is disrupted (e.g., after sectioning). Initially, the dye is weakly fluorescent, but it becomes more pronounced once incorporated into membranes.

Cy3 DIC18 is an orange-red fluorescent carbocyanine dye widely utilized as a lipophilic tracer. Its long hydrocarbon chains integrate into cell membranes, rendering it weakly fluorescent until incorporation. DiI diffuses laterally to stain entire cells, making it effective for anterograde and retrograde neuronal tracing. In intact tissue, minimal transfer occurs between labeled and unlabeled cells, though slight transfer may happen post-membrane disruption, such as during sectioning.