Immunohistochemistry Protocol

Immunohistochemistry is a technique that is commonly used in the localization and monitoring of proteins in tissue sections. This technique uses antibodies to analyze and detect proteins while maintaining the structure, cellular characteristics, and composition of native tissue. It is useful when used to assess and monitor treatment or progression of diseases. Also, the information that can be gathered through immunohistochemistry combined with microscopy helps provide an overall picture that allows researchers to make sense of the data they have obtained through other methods. In this technique, chemical fixation helps lock the molecular interactions in the cells. The tissue samples can be frozen, or paraffin-embedded before it is sectioned and mounted on slides for analysis. Depending on the sample of interest, the steps taken for collection, preservation, and fixation of the biospecimen can vary. 

Through specific antibody binding, immunohistochemistry helps to identify the pattern of protein expression. This bringing between antibody and epitope enables detection of particular amino acid sequences that are found within a protein. These antibodies can also be used to detect post-translational modifications. The tissue sample should be preserved and hardened to retain both form and structure so it can be sectioned. One crucial choice is deciding if immunohistochemistry should be carried out on fresh frozen samples or formalin fixed paraffin embedded (FFPE) samples. Immunohistochemistry on FFPE samples results in a superior cell or tissue morphology. However, one disadvantage would be the potential compromise of antigenicity by the fixation that is required. The antigenic site can be masked by the protein cross-links formed through formalin fixation. Antigen unmasking can be performed using enzymatic digestion (by pepsin, trypsin, or another protease) or heat (water bath, microwave, pressure cooker) with specific buffers (sodium citrate or ethylenediamine triacetic acid (EDTA)). 

Immunohistochemistry Protocol (for Formalin-Fixed Paraffin-Embedded Samples)

The basic steps include:

  • Fixation and embedding of the tissue

  • Cutting and mounting

  • Deparaffinization and rehydration

  • Antigen retrieval

  • Immunohistochemical staining

  • Counterstaining

  • Dehydration and stabilization

  • Examination of the staining 

A) Reagents and Solutions

  • Xylene

  • Ethanol – anhydrous denatured, histological grade (95% and 100%)

  • Hematoxylin

  • Wash buffer (10X Tris Buffered Saline with Tween (TBST)) – Prepare 1 liter of 1X TBST by adding 100ml 10 TBST and 900ml dH2O and mix

  • Antibody diluent options (SignalStain Antibody Diluent #8112, TBST 5% normal goat serum #5425, PBST 5% normal goat serum #5425)

  • Antigen unmasking options (Citrate: 10mM sodium citrate buffer, EDTA: 1mM EDTA, TE: 10mM Tris / 1mM EDTA, Pepsin)

  • 3% hydrogen peroxide

  • Blocking solution (TBST 5% normal goat serum)

  • Detection system (SignalStain Boost IHC Detection Reagents – Mouse #8125, Rabbit #8114)

  • Substrate (Signal Strain DAB Substrate Kit #8059)

B) Sample Preparation

  • Put the paraffin-embedded tissue in a mold along with a small amount of liquid paraffin. Cool momentarily to immobilize the tissue. Put the base of a cassette on the mold filling it with liquid paraffin. Cool. 

  • Cut the sample into thin sections (4 to 6μm) using a microtome and float the cut sections in a water bath. 

  • Mount the cut sections on charged slides and allow it to dry overnight. Charged slides are used as it helps the sections to adhere to the slide. 

C) Deparaffinization and Rehydration

Paraffin wax must first be removed from the sample followed by rehydration before antibody staining can be performed. It is important not to let the slides dry as it can result in inconsistent staining.

  • Remove the paraffin wax by placing the sections in 3 containers with xylene for about 5 minutes. Remember to use fresh xylene to avoid incomplete deparaffinization that can result in inconsistent staining. 

  • Rehydrate by placing the sections in 2 containers with 100% ethanol for 10 minutes. 

  • Place the sections in 2 containers that have 95% ethanol for 10 minutes.

  • Complete the rehydration process by washing the sections twice in dH2O for 5 minutes. 

D) Unmasking Antigens

Remove the cross-links formed during formalin fixation before performing antibody staining.

  • If using citrate – bring the slides to a boil in 10mM sodium citrate buffer at pH 6.0. Maintain it just below the boiling temperature for 10 minutes. Then, cool the slides on a bench top for approximately 30 minutes. 

  • If using EDTA – bring the slides to a boil in 1 mM EDTA at pH 8.0. Continue at a sub-boiling temperature for 15 minutes. The slides do not need to be cooled.

  • For TE – bring the slides to a boil in 10mM Tris and 1 mM EDTA at pH 9.0. Follow with a sub-boiling temperature for 18 minutes and cool for 30 minutes at room temperature. 

  • If using pepsin – digest at 37⁰C for 10 minutes.

E) Staining

The following protocol is for chromogenic staining. However, immunofluorescent staining can also be an option. 

  • First, wash the sections three times in dH2O for 5 minutes every time. 

  • Place the sections in 3% hydrogen peroxide for 10 minutes to stop endogenous peroxidase activity as this can cause high background staining. 

  • Again, wash the sections three times in dH2O for 5 minutes each. 

  • Then wash it in wash buffer for 5 minutes.

  • Taking care not to touch the sample, draw a large circle around the sample using a hydrophobic pen to create a hydrophobic boundary. This allows a smaller volume of antibody to be utilized while also allowing the staining using different antibodies on one slide.

  • Use 100 – 400 μl of blocking solution to block the sections in a humidified chamber for 1 hour at room temperature to avoid non-specific binding of the antibody to the tissues. 

  • Remove the blocking solution. Add 100 – 400 μl of primary antibody that has been diluted in the antibody diluent to each section. Incubate at 4⁰C overnight in a humidified chamber. Additional optimization may be necessary for shorter incubations.

  • Equilibrate the SignalStain Boost Detection Reagent until it reaches room temperature. 

  • Remove the antibody solution. Then, wash the sections in a wash buffer 3 times for 5 minutes each time.

  • Cover the sections with 1 to 3 drops of SignalStain Boost Detection Reagent and incubate these for 30 minutes in a humidified chamber at room temperature. 

  • Again, wash the sections 3 times in wash buffer for 5 minutes every time.

  • Add a drop of SignalStain DAB Chromogen Concentrate to 1 ml of SignalStain DAB Diluent. Mix before use.

  • Apply 100 – 400 μl of SignalStain DAB to every section and observe closely. Acceptable staining intensity is achieved in 1 to 10 minutes.  

  • Immerse the slides in dH2O.

  • An additional optional step is to counterstain the sections using hematoxylin. This will result in the staining of the cell nuclei and provide a contrast to the DAB chromogen to help with visualization of the tissue morphology.

  • Rewash the sections in dH2O twice for 5 minutes each. 

F) Dehydration and Mounting of Slides

SignalStain DAB chromogen is compatible with both nonaqueous and aqueous mounting mediums. If a nonaqueous medium is chosen, the sections have to be dehydrated again before mounting. 

  • Place the sections in 2 containers containing 95% ethanol for 10 seconds.

  • Then, place the sections in 2 containers with 100% ethanol for 10 seconds.

  • Next, place the sections in 2 containers containing xylene for 10 seconds. 

  • Mount the sections using coverslips with a mounting medium while avoiding the introduction of air bubbles. 

  • Allow the mounting medium to set.

  • View the slides on a microscope. 


Crosby K, Simendinger J, Grange C, Ferrante M, Bernier T, Standen C. Immunohistochemistry protocol for paraffin-embedded tissue sections. Cell Signaling Technology. Accessed 1/18/2019.