Immune-gold labelling for post-embedding
Embedding-medium options:
- LR White resin (Hard-grade Acrylic resin)
- Lowicryl HM20 non-polar and hydrophobic embedding medium (MonoStep kits)
- 2.3M Sucrose (or Gelatin – Sucrose/Methyl Cellulose)
Immune golds:
- Aurion NL
- BBI Solutions UK
Workflow for post-embedding at low temperature:
- Chemically fix tissue cells with 2% paraformaldehyde and 0.2% glutaraldehyde in NaHCa buffer
- Rapid-freeze by Leica EM HPM100 and freeze-substitute by Leica EM AFS2-FSP (as required)
- Infiltrate in a variety of resin/acetone mixtures
- Polymerise in LR White at 4°C or Lowicryl HM20 at -60°C under UV irradiation by Leica EM AFS2
- Ultra-section at RT and mount with the carbon-coated Formvar membrane on Nickel or Gold grid
- Rinse with the buffer
- Quench with 50mM NH4Cl, 50mM Glycine, 50mM Lycine (which is done on the surface of 100 µl drops x 7 from this procedure)
- Wash with the buffer
- Block with BSA and normal serum mixtures in the buffer solution
- Immune-label with primary antibody in 0.1% BSA-containing buffer
- Wash with 0.1% BSA-containing buffer
- Immune-label with secondary antibody-conjugated colloidal golds in 0.1% BSA-containing buffer
- Wash with the buffer
- Glutar-fixation and wash with water
- Grid stain with uranyl acetate or UA-Zero
- Wash with water and natural dry
- TEM imaging
Workflow for Tokuyasu method:
- Chemically fix tissue cells with 2% paraformaldehyde and 0.2% glutaraldehyde in NaHCa buffer
- Wash with the buffer
- Quench
- Infiltrate with gelatin, and then with sucrose
- Mount on the specimen pins for cryo-ultramicrotomy
- Vitrification
- Cryo-trim the frozen block with Diatome cryo-trim diamond knife (35°) by Leica EM UC7-FC7 at liquid nitrogen temperature
- Cryo-ultra-section with Diatome cryo-immune diamond knife (35°), and ribbon collection with Leica EM double-manipulator at liquid nitrogen temperature
- Pick up the cryo-section on the grid by Leica EM Crion
- Immuno-gold labelling