Visualizing nuclei in skin cryosections: viable options to 4'6-diamidino-2-phenylindol for confocal laser microscopy.
BACKGROUND
Visualization of nuclei in skin (cryo-) sections is essential for both, rapid overview and reliable orientation within skin samples. Therefore, nuclear staining is a very common counterstain for immunohistochemical studies of human skin as this nuclear staining precisely depicts the cellular distribution within the epidermis. Moreover, it clearly shows the epidermal-dermal border as well as the transition zone between the living and the cornified layers of the epidermis. For standard epifluorescence microscopy, 4'6-diamidino-2-phenylindol (DAPI) is commonly used. For confocal laser scanning microscopy (CLSM), however, DAPI is often not suitable because its excitation maximum is in the ultraviolet (UV) range (Ex(max) 359 nm) when bound to DNA, and UV lasers and the corresponding optics are not part of CLSM standard configuration.


METHODS
In order to find an adequate DAPI substitute that is excitable with standard visible light lasers, the following nuclear stains were tested: LOLOt-1 iodide (Ex(max) 565 nm), TOTO s -3 iodide (Ex(max) 642 nm), LO-PROt-1 iodide (Ex(max) 567 nm), SYTO s 84 (Ex(max) 567 nm), SYTO s 85 (Ex(max) 567 nm), SYTOX s Green (Ex(max) 488 nm) and SYTOX s Orange (Ex(max) 547 nm), Propidium iodide (Ex(max) 535 nm). Besides optimal concentration and incubation time, following criteria were also evaluated: photobleaching, background, e.g. cytoplasmic staining of RNA, and sensitivity to different fixation conditions (unfixed, IEM fixation, PLP fixation and PFA fixation).


RESULTS
According to these criteria Sytox s Green showed the best overall staining score and can be used for variously fixed skin samples and shows a distinct and stable green nuclear fluorescence.
Gläser
K
K
4226 Advanced Development Deo/AP, Beiersdorf AG, Hamburg, Germany.
Wilke
K
K
Wepf
R
R
Biel
S S
SS
D002467:Cell Nucleus / Q000648:ultrastructure*; D003287:Contrast Media; D016616:Cryoultramicrotomy; D046169:Dermoscopy / Q000379:methods*; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009930:Organic Chemicals; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000166:cytology*
D003287:Contrast Media; D009930:Organic Chemicals; C402795:SYTOX Green