1. Alcian Blue – Nuclear fast red
(Kernechtrot)
Stain design
properties
polyvalent; copper
anionic; aluminium
colour
blue
red
counterstain
acid mucopolysaccharides
and acetic mucins.
specificity
calcium
nuclei : pink to red,
tissue elements pale pink to red

2. Alcian Blue – Nuclear fast red
Stain design
pH pH 2.5 (3% acetic acid solution)
sulphated and non-sulphated acidic carbohydrates pH
pH 1.0
only sulphated carbohydrates
composition - 3% acetic acid solution………3 min
- 1% Alcian Blue solution…….30 min
- 0,1% Nuclear fast red………..5 min
fixation formalin fixed, paraffin embedded tissue sections.
procedure manual; 1h 25min

3. Alcian Blue – Nuclear fast red
(pH2,5)
Stain target
generic nuclei : red
background: pale pink
bright blue: sulfated and carboxylated acidic mucopolysaccharides
weakly sulphated mucins and sulphomucins,
hyaluronic acid
sulfated and carboxylated sialomucins (glycoproteins)

4. Alcian Blue – acidic mucosubstances
Stain target
Choanocytes
central flagellum
collar of microvilli
thin membrane
‘Verongia sp.’ (Fixation for FISH)

5. Alcian Blue – acidic mucosubstances
Stain target
pinacoderm
(thin epithelial layer - glycoprotein)

6. Nuclear fast red – nuclei
Stain target
Choanocytes
Embryonic matrix?
Mobile sponge cells
Rogue, PJ, Malviya, AN (1999) Calcium signals in the cell nucleus. The EMBO Journal18:

7. Nuclear fast red – cytoplasm
Stain target
Spongin
Bacteria ?
Sponge mesohyl

8. Alcian Blue – Nuclear fast red
Stain target
bacteria
Cyanobacteria

9. Alcian Blue stains bacterial capsules and insoluble polysaccharides
Alcian Blue – Nuclear fast red
Bacteria
stain
Bacterial cell wall
No diffusion through cell wall X
Bacterial protoplasm
Lack of internal polysaccharides?
Alcian Blue 8GN150 dye was found to stain
the bacterial capsules and insoluble polysac-
charides in both puire and mixed cultures of
bacteria andlprotozoa. This dye was found to be
specific for any insoluble carbohydrate. At acid
pH there was an affinity for acids as well as
carbohydrates. The reaction was believed to be
due to salt linkage in the case of the acids and
to the internal hydroxyl group in the case of
carbohydrates. The fact that bacterial protoplasm
did not stain might be attributed to the complex-
ity of the dye molecule preventing diffusion
through the cell wall rather than the lack of
affinity for the internal polysaccharides for the
stain. R
Ross E. McKinney (1953) Staining bacterial polysaccharides. J Bacteriol 66(4): 453–454.
Alcian Blue stains bacterial capsules and insoluble polysaccharides 9

10. Alcian Blue – Nuclear fast red
(pH2,5)
Stain target
specific
(Verongia sp) red: nuclei, spongin, embryonal matrix
pale pink: sponge mesohyl
bright blue: flagellum/microvilli (mucus);
epithelial glycoprotein,
bacterial capsules and insoluble carbohydrates
(acidic mycopolysaccharides and mucins)

11. Alcian Blue – Nuclear fast red
Stain efficiency
CCChhhrrrrrrr!!!!