Toluidine 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

0.1M acetate buffer

13.6g anhydrous sodium acetate

900ml dH2O

pH to between 3.75 and 4.25 using glacial acetic acid

Toluidine Blue

0.04g Toluidine Blue

100ml 0.1M sodium acetate buffer (pH 3.75-4.25)

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!)

.                    ↓

Toluidine Blue 10min

.                    ↓

dH2O 2x 5min

.                    ↓

Fast Green/Fast Red 3-5min

.                    ↓

dH2O 2x 5min

.                    ↓

95% EtOH 3min

.                    ↓

100% EtOH 5min

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

End result: nuclei red, proteoglycans purple