Comparison of immunogold staining after self-pressurised rapid freezing, high pressure freezing and classical chemical fixation. This is an example of our method to measure the density of immonlabeling, see this page
For the fixation of SPRF and HPF, see Claeys et al. 2017 Nematology. For the chemical fixation:
- Primary fixation : 30 min at 4°C in 2,5% glutaraldehyde in 0,05 M sodium cacodylate buffer (pH 7,4) with 0.25 mg/ml MgCl2;
- cut nematode at the head and tail region (to improve the impregnation);
- Overnight fixation in fresh portion of glutaraldehyde fixer (4°C, rotation);
- Rinsing 4 times (5 min, 15 min, 1h, 1h (or overnight at 4°C, rotation) in buffer;
- Post fixation: 2 hours in 2% osmium tetroxide in the same buffer (room temperature, rotation);
- Rinsing in AD 3 times 20 min (room temperature);
- en bloc for 1h in 1% solution of uranyl acetate in distilled water (in darkness, room temperature, rotation);
- dehydrated in ethanol series followed by a propylenoxide series (20 min each step);
- impregnation and embedding in low viscosity embedding medium Spurr resin (EMS, Hatfield, UK).
