Frozen Sectioning & Immunogold

Frozen Sectioning & Immunogold

Immunogold Labeled Endoplasmic reticulum

Preparation of ultrathin frozen sections (Tokuyasu)

Fixation of cells in culture: Attached cells: first remove cells from the dish with 5mM EDTA/PBS using a transfer pipet with a wide opening. (You can use trypsin if you are not interested in surface proteins). Pipet up and down to release the cells from the dish as quickly as possible. Add cell suspension to a microfuge tube containing an equal volume 2x fixative (8% formaldehyde) in 0.2 M sodium phosphate buffer pH 7.4. (not PBS) and spin down to a pellet (~3000rpm for 3 min). Take off supernatant and add layer fresh 4%formaldehyde in 0.1M Sodium Phosphate buffer on top of the pellet - do not resuspend the cells. Leave pellet to fix for 2 hrs at RT. It's important not to resuspend the cells after they have been pelleted, this way the pellet will crosslink and stay together better in the sucrose infiltration step.

Cryoprotection: Small pieces (~2mm) of tissue or cell pellet is placed in a drop of 2.3 M Sucrose in PBS containing 0.15M glycine. Move to a fresh drop after a few minutes, do 3 drops for a total of 15 min at RT (or overnight at 4 °C).

Freezing: Sucrose infiltrated tissue/cells are mounted on an aluminum pin, excess sucrose is removed with a filterpaper and the pin is plunged into liquid nitrogen.  Specimens can be stored for years under liquid nitrogen in cryo vials.

Sectioning: Ultrathin sections are cut at -120°C with a cryo-diamond knife, or at -90°C with a glass knife. Sections are picked up from the knife with a loop dipped a mixture of  2.3M sucrose and 2% Methylcellulose (I usually use 9 parts sucrose to 1 part MC) and transferred to a formvar/carbon coated copper grid. Grids are left floating section side down on PBS or placed on 2% gelatin in a small petri dish and stored in the fridge until the immunogold labeling can be done.

Immunogold labeling procedure for Ultrathin Frozen Sections

If you stored the sections on 2% gelatin, warm up gelatin to 37degrees for 20 minutes, the gelatin will melt and you can transfer the grids to drops of PBS. Wash on 4 drops of PBS before blocking in 1% BSA.

  1. Blocking, 1% BSA in PBS (15 min)
  2. 1° Antibody diluted in 1%BSA (30 min RT)
  3. Wash, 4 drops PBS (15 min)
  4. If 1° antibodies with weak binding capacity for protein A (mouse, rat, goat, sheep) are used you need to use a bridging antibody (e.g. rabbit anti mouse) (30 min) followed by washing in 4 drops of PBS before the Protein A-gold step.
  5. Protein A-gold (PAG) in 1% BSA (20 min)
  6. Wash, 4 drops PBS (15 min)
    In case of double labeling, fix in 1% Glutaraldehyde for 5 min, followed by quenching in 4 drops of 0.15 M Glycine/PBS and repeat points 2-7 with another size PAG.
  7.  Wash, 4 drops PBS (15 min)
  8. Wash, 6 drops water (20 min)

Contrasting procedure: Mix 9 parts 2% methyl cellulose with 1 part 3% aqueous uranyl acetate. Float grids on the mixture on ice for 10 min. Pick up with a 3.5mm loop and remove excess liquid with a filterpaper (Whatman #1). Allow to dry before gently removing the grid from the loop and examining in the TEM