Electron microscopy of cartilage - sample prep

Sample preparation

sacrifice the animals up to 1 week of age and dissect knee samples

Procedure

fix in freshly made up 2.5% glutaraldehyde in 0.1M sodium cacodylate (pH7.3, 2h+) at 4°C

.                    ↓

wash 3x 10min in 0.1M sodium cacodylate at 4°C

.                    ↓

fix in 2% OsO4 in 0.1M sodium cacodylate ( in distilled water mixed in a 1:1 ratio with 3% potassium ferrocyanide in 02M sodium cacodylate buffer, 2h) at room temperature

.                    ↓

wash 3x 10min in ddH2O at room temperature

.                    ↓

stain overnight in 2% aqueous uranyl acetate 2h / 0.5% uranyl acetate

.                    ↓

wash 3x 10min with ddH2O at room temperature

.                    ↓

dehydrate in increasing concentrations of acetone (50%, 70%, 90%) 30 min each

.                    ↓

incubate 3x 30min in 100% acetone

.                    ↓

2x 5min propylene oxide

.                    ↓

2:1 ratio of propylene oxide:TAAB LVEM resin 2h at room temperature, on a rotator

.                    ↓

1:1 ratio of propylene oxide:TAAB LVEM resin 2h at room temperature, on a rotator

.                    ↓

1:2 ratio of propylene oxide:TAAB LVEM resin overnight at room temperature, on a rotator

.                    ↓

100% resin (3 changes) over 2 days, on a rotator

.                    ↓

place in a flat mold and polymerise overnight at 60°C for 1-2 days

.                    ↓

remove frm molds and section

Note

The samples will have to be counterstained with 0.3% led citrate.

end result: