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Immune-gold labelling for post-embedding

 

Embedding-medium options:

  1. LR White resin (Hard-grade Acrylic resin)
  2. Lowicryl HM20 non-polar and hydrophobic embedding medium (MonoStep kits)
  3. 2.3M Sucrose (or Gelatin – Sucrose/Methyl Cellulose)

 

Immune golds:

  1. Aurion NL
  2. BBI Solutions UK

 

Workflow for post-embedding at low temperature:

  1. Chemically fix tissue cells with 2% paraformaldehyde and 0.2% glutaraldehyde in NaHCa buffer
  2. Rapid-freeze by Leica EM HPM100 and freeze-substitute by Leica EM AFS2-FSP (as required)
  3. Infiltrate in a variety of resin/acetone mixtures
  4. Polymerise in LR White at 4°C or Lowicryl HM20 at -60°C under UV irradiation by Leica EM AFS2
  5. Ultra-section at RT and mount with the carbon-coated Formvar membrane on Nickel or Gold grid
  6. Rinse with the buffer
  7. Quench with 50mM NH4Cl, 50mM Glycine, 50mM Lycine (which is done on the surface of 100 µl drops x 7 from this procedure)
  8. Wash with the buffer
  9. Block with BSA and normal serum mixtures in the buffer solution
  10. Immune-label with primary antibody in 0.1% BSA-containing buffer
  11. Wash with 0.1% BSA-containing buffer
  12. Immune-label with secondary antibody-conjugated colloidal golds in 0.1% BSA-containing buffer
  13. Wash with the buffer
  14. Glutar-fixation and wash with water
  15. Grid stain with uranyl acetate or UA-Zero
  16. Wash with water and natural dry
  17. TEM imaging

 

Workflow for Tokuyasu method:

  1. Chemically fix tissue cells with 2% paraformaldehyde and 0.2% glutaraldehyde in NaHCa buffer
  2. Wash with the buffer
  3. Quench
  4. Infiltrate with gelatin, and then with sucrose
  5. Mount on the specimen pins for cryo-ultramicrotomy
  6. Vitrification
  7. Cryo-trim the frozen block with Diatome cryo-trim diamond knife (35°) by Leica EM UC7-FC7 at liquid nitrogen temperature
  8. Cryo-ultra-section with Diatome cryo-immune diamond knife (35°), and ribbon collection with Leica EM double-manipulator at liquid nitrogen temperature
  9. Pick up the cryo-section on the grid by Leica EM Crion
  10. Immuno-gold labelling

Group Members

Jaime Llodra Gonzalez

EM Research Specialist

jl2235@mrc-tox.cam.ac.uk

 

Maria Guerra Martin

Senior Research Officer

mag88@mrc-tox.cam.ac.uk

 

Nobuhiro Morone

Senior Investigator Scientist

Facility Head

nm669@mrc-tox.cam.ac.uk