Chemistry Reference
In-Depth Information
Triphenylmethane
Triarylmethine dyes
λ
abs
(nm)
λ
em
(nm)
Remarks
Methyl violet dyes
580-590
~ 590
/
Fuchsine dye
547-552
625
50% aqueous EtOH
Malachite green
446
614
/
Aniline blue
600
Mixture of methyl blue and water blue
fIgure 11.8
The basic skeleton of staining agents belonging to the triarylmethine dye family.
O
8
1
7
2
3
HO
OH
Alizarin: 1,2-Dihydroxyanthraquinone
(λ
abs
= 438 nm, 450-500 nm after complexed with Ca
2+
)
6
4
5
O
fIgure 11.9
Dihydroxyanthraquinone dyes.
table 11.1
Various azo and diazo staining agents.
Important azo- and diazo staining agents
λ
abs
(nm)
λ
em
(nm)
remarks
Janus Green B
630
50% aqueous EtOH
Congo red
497
614
614 nm upon binding to amyloid
Sudan II
490
EtOH
Sudan III
503
EtOH
Sudan IV
520
EtOH
Oil red O
518
toluene
Sudan Black B
598
EtOH
Tryphan Blue
588
600-670
600-670 when bound to proteins
Evans Blue
611
680
/
11.3.1.4 dihydroxyanthraquinones
The first dihydroxyanthraquinone dye, Alizarin (Figure 11.9), was introduced as a
staining agent in 1874. It is still being used nowadays to reveal calcium in tissues and cells because it forms a distinct red
precipitate with calcium ions [42, 43].
11.3.2
azo- and diazo-staining agents
Toward the late 19th to early 20th century, other synthetic organic dyes began to emerge as useful histological staining
agents. Amongst them are the azo- and diazo-dyes, and the reducible tetrazolium probes. Commonly used azo- and diazo-
dyes in microscopy staining include Janus Green B, Congo red, the Sudan lysochromes (Sudan II, III, and IV, Sudan Black B,
and Oil red O dyes, Table 11.1) and Trypan and Evans Blue. Janus Green B is a mitochondria-specific stain [43-45]. Congo
red is used as a cytoplasm and erythrocyte stain, as well as for the detection of amyloid plaques resulting from Alzheimer's
disease [46, 47]. Besides being a colorimetric dye, Congo red can also fluoresce to be used as a fluorescent stain for elastin
[48] and polysaccharides [49-52].
The diazo dyes Trypan and Evans Blue were introduced as cell and tissue vitality stains, via exclusion, in 1915 [53, 54].
Both of them are colorimetric as well as fluorescent dyes. Most viable cells and tissues are able to exclude them, while the
proliferated plasma membrane of dead cells cannot stop their penetration. Hence, they are used to stain the dead cells. Such
an exclusion mechanism is different from the metabolic assessment mechanism of the MTT cell viability test using the
tetrazolium compound methylthiaolyldophenyl tetrazolium (MTT) (to be discussed later). Tryphan Blue can also bind to
proteins, especially albumin, give distinct red fluorescence at 600-670 nm, and is used to study exudation from blood vessels
