Alcian Blue staining

Sample preparation

1. sacrifice the animals and dissect knee samples
2. fix in 4%PFA over 48h in the fridge
3. decalcify in 20% EDTApH7.4 for 2 weeks (shaking)
   
4. wax embed and section (6um sections)

Note

to generate reliable data, this experiment should be performed on matched sections from 3 unrelated animals per genotype

Alcian Blue

15mg Alcian Blue

80ml 95% EtOH

20ml glacial acetic acid

adjust pH to 0.75

Note

pH 0.75 ensures Alcian Blue specificity to sulphated proteoglycans and is a recommended pH for staining cartilage. At pH 2.5 it stains most acid mucins (except some of the strongly sulphated groups), at pH 1.0 it will stain only weakly and strongly sulphated acid mucins, and at pH 0.2 it will only stain strongly sulphated acid mucins.

Staining steps

dewax in xylene 2 x 5min

.                    ↓ 

100% EtOH 3min

.                    ↓

90% EtOH 3min

.                    ↓

70% EtOH 3min

.                    ↓

50% EtOH 3min

.                    ↓

dH2O 2x 3min (from here on, do not let the samples dry!)

.                    ↓

Alcian Blue 10min

.                    ↓

dH2O 2x 5min

.                    ↓

Fast Red 5min

.                    ↓

dH2O 2x 5min

.                    ↓

95% EtOH 3min

.                    ↓

100% EtOH 5min

.                    ↓
dehydrate in xylene 2x 5min
.                    ↓
coverslip, dry overnight

End result: nuclei red, sulphated proteoglycans blue