Immunofluorescence

IF on OCT frozen sections

  • Blocking media: PBS/0.1%Tw20/2.5%BSA/5% Goat Serum
  • Anti-fade mounting medium (ProLong® Gold, Invitrogen, Cat# P36931)
  • Hoechst 33342 (10 mg/ml stock; use at 1/10,000; Invitrogen)
  1. Dry slides for 10 min on slide dryer
  2. Fix with 100% acetone for 10 min at -20°C. Air dry for ~10 min. Draw boundaries with PAP pen.
  3. Wash 2 times for 3 minutes each with PBS
  4. Block for 1 hr with blocking buffer
  5. Dilute primary antibodies in blocking buffer and incubate tissue in 200 ml for 1.5 hr at RT or O/N at 4°C.
  6. Wash 3x 5min with PBS
  7. Re-block for 10 min
  8. Dilute secondary antibodies in blocking buffer and incubate for 1 hr at RT.
  9. Wash 1x 5min in PBS
  10. Add Hoeschst 33342 at 1/10,000 for 15 min.
  11. Wash 3x 5 min in PBS
  12. Carefully mount tissue on glass slides with ~2 drops of Prolong Gold mounting medium. Incubate overnight at RT in the dark to cure.
  13. Seal the edges with nitrocellulose based lacquer (nail polish)
  14. Mounted tissue should be stored protected from light at 4ºC and should be imaged as soon as possible to obtain best imaging results
  15. Analyze

IF on FFPE Sections

(A) Deparaffinize slides: Xylene (2x5 min), 100% EtOH (2x3 min), 95% EtOH (1 min), 80% EtOH (1 min), ddH2O (≥ 5 min)

(B) Staining

  1. Antigen retrieval (select one)
    1. EDTA buffer
      • 1 mM EDTA + 0.05% Tween 20 in DI water, pH 8.0
      • Immerse slides in EDTA buffer in slide container
      • Place slide container in a steamer for 30 minutes
      • Cool for 30 minutes in the same buffer
    2. Citrate buffer
      • 10 mM sodium citrate + 0.05% Tween 20 in DI water, pH 6.0
      • Immerse slides in citrate buffer in slide container
      • Place slide container in steamer for 30 minutes
      • Cool for 30 minutes in the same buffer
    3. Proteinase K
      • Apply Proteinase K solution for 30 s
      • Rinse slides
  2. Wash 2x5 minutes in PBS
  3. Using PAP pen, draw around sections
  4. Block the section for 1 hour in blocking buffer (5% fetal horse serum, 1 mg/mL BSA, 0.05% Tween 20, 1X PBS) in a humidified chamber at RT
  5. Remove the blocking buffer
  6. Add the primary antibody, diluted to the desired concentration in blocking buffer. Incubate 1.5 hours at RT in humidified chamber (or overnight at 4°C)
  7. Remove the primary antibody
  8. Wash slides 3x 5 minutes in 1x PBS
  9. Re-block for 10 min
  10. Dilute secondary antibodies in blocking buffer and incubate for 1 hr at RT.
  11. Wash 1x 5min in PBS
  12. Add Hoeschst 33342 at 1/10,000 for 15 min.
  13. Wash 3x 5 min in PBS
  14. Carefully mount tissue on glass slides with ~2 drops of Prolong Gold mounting medium. Incubate overnight at RT in the dark to cure.
  15. Seal the edges with nitrocellulose based lacquer (nail polish)
  16. Mounted tissue should be stored protected from light at 4ºC and should be imaged as soon as possible to obtain best imaging results
  17. Analyze

Note: FFPE tissues have higher background in the green channel. A highly expressed antigen or use of a Brilliant Blue 515 secondary is recommended to compensate for this.