Immunohistochemistry with TSA

Contributed by Luke Hammond, QBI, The University of Queensland, Australia

Immunohistochemistry with Tyramide Signal Amplification (TSA) using Perkin Elmer kit & Protocol for Vibratome Sections

Perform all steps with slides arrayed horizontally in humidified chamber on a rotator at RT.


Sodium citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20 pH 6) 2.94g Tri-sodium citrate // 1000ml DI water // adjust pH to 6.0 with 1N CL // add tween 20 // mix well // store at room temperature for 3months or at 4ºC for longer storage 50 ml of 1xPBS pH 7.4 50ml of 1% BSA/PBS


  • Mount sections on SuperFrost Plus slides – dry for ~1hr to fully adhere sections to slide
  • Rinse slide with PBS to rehydrate
  • Fix in 4% PFA for 10min
  • Wash 3x 3min in PBS
  • Perform antigen retrieval in Biocare Medical decloaking chamber – heat sections to 125oC for 4min at 15 psi in sodium citrate buffer
  • Wash 3x 3min in PBS
  • Block sections in 0.9% H2O2/10% Normal Serum/0.2% Triton-X 100 in PBS for 2hr on rotator (serum should be same as the species from which your secondary antibody was generated)
  • Incubate overnight with 1o antibody/ies at desired concentration in 2% Normal Serum/0.2% Triton-X 100 in PBS
  • Wash 3x20min in PBS
  • Incubate with appropriate HRP labelled IgG 2o antibody diluted 1:500 in 0.2% Triton-X 100 in PBS for 1hr
  • Wash 3x10min in PBS
  • Dilute TSA Amplification reagent 1:50 in 1xPlus Amplification Diluent buffer.
  • Apply 150-200µL per slide and incubate for 1 – 10mins (need to optimize for individual antibodies) in humidified chamber covered in foil. For all subsequent steps, keep chamber covered in foil
  • Wash 3x10min in PBS

If performing double labelling for multiple primary antibodies proceed to step 15. If only performing single labelling with TSA kit proceed to step 16 Incubate with appropriate fluorescent 2o antibody diluted in 0.2% Triton-X 100 in PBS for 3hr.

  • Incubate in DAPI diluted 1:1000 in 0.2% Triton-X 100 in PBS for 20min at RT.
  • Wash 3x20min PBS. NB: If using 2o antibodies at high concentrations (i.e. greater than 1:500) replace first two washes with 0.2% Triton-X 100 in PBS.
  • Coverslip using ProlongGold (Invitrogen). Let mounting media set for 1hr at RT in slide book, then store at 4oC in dark. Take images within 2-3 weeks for optimum fluorescence.

This method is based, with permission, on an original protocol available here.