We use Immunocytochemistry to investigate protein-complexes.
Acrylic resins:
1) LR White.
2) Lowicryl K11M.
Workflow – 1:
1) Chemically fix tissue cells with paraformaldehyde solution.
2) Infiltrate in a variety of resin/acetone mixtures.
3) Polymerise in LR White at -20°C or Lowicryl K11M at -60°C under UV irradiation.
4) Cut the ultra section at room temperature.
5) Block with BSA buffer solution.
6) Immuno label with 1° antibody & 2° antibody-conjugated colloidal golds on the grid.
7) Chemically fix with glutaraldehyde and wash with water.
8) TEM.
Workflow – 2:
1) Vitrificate tissue cells by HPF.
2) Place the cells in cold uranyl acetate in methanol at -90°C for several hours – days.
3) Follow same procedure as for Freeze substitution.
4) Embed cells in Lowicryl K11M and polymerise at -60°C by UV irradiation.
5) Cut the ultra section at room temperature.
5) Block with BSA buffer solution.
6) Immuno label with 1° antibody & 2° antibody-conjugated colloidal golds on the grid.
7) Chemically fix with glutaraldehyde and wash with water.
8) TEM.
Post-embedding immuno-gold labelling
Lung carcinomas