Immunolabeling Techniques

Contact: Bobbie Schneider
Contact phone: (206) 667-4289

Training Type: Self-Directed
Training Format: How-to

Procedure for Post-embedding single labeling:

  1. Block free aldehyde groups with 50mM glycine in PBS for 20 min.
  2. Wash with PBS (3 drops) quickly
  3. Block with 5% BSA in PBS (1 drop) for 30 min.
  4. Wash in incubation buffer - BSA-c buffer (PBS+0.1%BSA-c,pH 7.4)/tween 20 (if needed for background) 6x2 min. each.
  5. Primary antibody incubation: Incubate with a dilution of specific primary antibody, preferably Affinity-purified, 1-5 ug/ml in incubation buffer, for 30 min-I hour at room temperature. If longer incubation times are required, carry out the procedure at 4 degrees C overnight.
  6. The grids are washed on drops of incubation buffer for 6 x 5 min. each
  7. Incubate with gold conjugate reagents, 1/20-1/40 in incubation buffer For 1-2 hrs
  8. Wash grids on drops of incubation buffer for 6 x 5 min. each
  9. Wash grids on drops of PBS for 4 x 5 min. each
  10. Post fix in 2% glutaraldehyde in PBS for 10 min at room temp.
  11. Wash grids in distilled water with wash bottle.
  12. Contrast with UA and lead

Procedure for Pre-embedding single labeling:

  1. Block free aldehyde groups in 0.1% NaBH4 in 0.1M PB for 10 min.
  2. Rinse in 0.1M PB, 4 x5 min.
  3. Permeabilization: (optional)
  4. Treat samples with 0.05% Triton-x-100 in PB
    For Cultured cells: 10-15 min.
    For Vibratome sections: 30 min.
  5. Wash in PB 3 x 10 min.
  6. Block Step:
    Block in Aurion Blocking Solution for 30 min.-1 hr.
  7. Wash Steps:
    Wash in BSA-c buffer 3 x 10 min.
  8. Primary Antibody Incubation:
    Incubate in Primary (1-5 ug/ml) diluted in BSA-c buffer overnight at
    4 degrees C preferable.
    Negative controls remain on BSA-c
  9. Wash Steps:
    Wash with incubation buffer 6 x 10 min.
  10. Immunogold Conjugate:
    Incubate with the secondary gold conjugate reagent in incubation buffer
    Preferable over night at 4 degrees C or at least 4 hr.
  11. Wash Steps:
    Wash with incubation buffer, 6 x 10 min.
    Wash with PBS, 6 x 5 min.
  12. Postfix in 2% glutaraldehyde in 0.1M PB, 30 min.
    Wash in 0.1M PB for 2 x 5 min.
    Wash in distilled water for 5 x 30 sec.
  13. Optionally:
    Osmicate in 0.5% OsO4 in PB for 15 min. and then wash in PB and further process.