IHC Standard Protocol

The protocol described below is the Atlas Antibodies standard immunohistochemistry protocol optimized for Triple A Polyclonals and PrecisA Monoclonals.


Download a pdf version of the standard immunohistochemistry protocol.



Paraffin sections of 4 µm thickness are baked overnight at 50°C. Prior to immunostaining, deparaffinization and hydration are performed in xylene and graded ethanol to distilled water. During hydration, we perform 5 minutes blocking for endogenous peroxidase in 0.3% H2O2 in 95% ethanol. 

Antigen retrieval

1. Standard antigen retrieval method 

The standard antigen retrieval method is Heat Induced Epitope Retrieval (HIER) in retrieval buffer pH 6.1 (Dako, Agilent, Santa Clara, CA, USA) using a pressure boiler (Decloaking chamber NxGen, Biocare Medical, Pacheco, CA, USA) as heat source.

HIER is performed by heating the TMA-slides immersed in retrieval buffer for 20 minutes at 110°C in the pressure boiler. After completing the boiling, slides remain in the pressure boiler and are allowed to cool to 90°C. The total processing time is approximately 60 minutes.

NOTE: The specified working dilutions of the primary antibodies are to be considered as a guideline only. Optimal dilutions must be determined by the user.

2. Alternative antigen retrieval methods

For selected antibodies, alternative retrieval buffers and/or enzymatic antigen retrieval may be used as stated in the Product Datasheet and on the Antibody/ Antigen information page on the Human Protein Atlas website.

Enzymatic antigen retrieval method 
Enzymatic retrieval is performed in the immunostaining instrument by incubating TMA-slides in Proteinase K solution (Lab Vision, Freemont, CA, USA) for 10 minutes at room temperature.

Heat-Induced Epitope Retrieval (HIER) in Retrieval Buffer pH 9
HIER is performed in Target Retrieval solution pH 9 (Dako, Agilent, Santa Clara, CA, USA) using Decloaking Chamber NxGen as described above. 

Immunohistochemical staining program

All incubations are performed at room temperature.
All reagents are applied at a volume of 300 µl per slide.

  1. Rinse in wash buffer.*
  2. Incubate with Ultra V Block for 5 minutes.
  3. Rinse in wash buffer (x2).
  4. Incubate with primary antibody for 30 minutes.
  5. Rinse in wash buffer (x3).
  6. Incubate with Primary Antibody Enhancer for 20 minutes.**
  7. Rinse in wash buffer (x2).
  8. Incubate with labeled polymer for 30 minutes.
  9. Rinse in wash buffer (x2).
  10. Develop in DAB solution for 5 minutes.
  11. Rinse in distilled water.
  12. Counterstain in hematoxylin for 5 minutes.***
  13. Rinse in tap water for 5 minutes.
  14. Rinse in lithium carbonate (Li2CO3) water, diluted 1:5 from saturated solution for 1 minute.
  15. Rinse in tap water for 5 minutes.
  16. Dehydrate in graded ethanol and xylene
  17. Mount in Pertex.
  18. Coverslip.

* Steps 1 – 11 are performed in Autostainer 480S (ThermoFisher Scientific, Waltham, MA, USA).

** For polyclonal antibodies skip steps 6 and 7.

*** Steps 12 -18 are performed in fully automated integrated stainer Leica ST5010-CV5030 (Leica Biosystems Nussloch GmbH, Nussloch, Germany).


For immunohistochemistry, the following reagents are commercially available from Thermo Fisher Scientific, Waltham, MA, USA:

  • Wash buffer (10x concentrate). Working solution originally contains 0.05% (v/v) Tween 20. Extra Tween 20 is added to a final concentration of 0.20%.
  • Antibody diluent
  • UltraVision LP HRP polymer®
  • Primary Antibody Enhancer (only for monoclonal antibodies)
  • Ultra V Block
  • DAB Quanto Detection System (including chromogen and substrate)

For epitope retrieval, the following buffers are commercially available from Dako, Agilent, Santa Clara, CA, USA:

  • Target Retrieval Solution, Citrate pH 6.1 (10x)
  • Target Retrieval Solution, pH 9 (10x)

In addition, Mayer’s hematoxylin and xylene (Histolab, Gothenburg, Sweden) are used.