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The IHC-plus™ Immunohistochemistry Protocol

LSBio has tested and validated monoclonal antibodies (mouse, rabbit, rat), polyclonal antibodies (rabbit, goat, sheep, llama, chicken), and human single and double chain antibodies for use in immunohistochemistry (IHC) using formalin-fixed paraffin-embedded tissues, the most common fixation method used by pathology labs worldwide. After 15 years of experience performing contract IHC research, creating immunohistochemistry localization databases, and producing and testing antibodies in IHC, we have acquired a substantial body of experience regarding IHC and what protocol works with the highest percentage of commercial antibodies and available tissues. Here is our protocol for validating IHC-plus antibodies. 

Tissue Preparation

  • Formalin fixation and embedding in paraffin wax

Tissue Sectioning

  • Make 4-µm sections and place on pre-cleaned and charged microscope slides.
  • Heat in a tissue-drying oven for 45 minutes at 60°C


  • Wash slides in 3 changes of xylene – 5 minutes each at room temperature.


  • Wash slides in 3 changes of 100% alcohol – 3 minutes each at room temperature.
  • Wash slides in 2 changes of 95% alcohol – 3 minutes each at room temperature.
  • Wash slides in 1 change of 80% alcohol – 3 minutes at room temperature.
  • Rinse slides in gentle running distilled water – 5 minutes at room temperature.

Antigen retrieval

  • Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C - 20 minutes
  • Remove from heat and let stand at room temperature in buffer - 20 minutes
  • Rinse in 1X TBS with Tween (TBST) – 1 minute at room temperature.


  • Do not allow tissues to dry at any time during the staining procedure.
  • Apply a universal protein block – 20 minutes at room temperature.
  • Drain protein block from slides, apply diluted primary antibody – 45 minutes at room temperature.
  • Rinse slides in 1X TBST - 1 minute at room temperature.
  • Apply a biotinylated secondary antibody (specific to the host of the primary antibody) - 30 minutes at room temperature.
  • Rinse slides 1X TBST – 1 minute at room temperature.
  • Apply alkaline phosphatase streptavidin – 30 minutes at room temperature.
  • Rinse slides in 1X TBST - 1 minute at room temperature.
  • Apply alkaline phosphatase chromogen substrate – 30 minutes at room temperature.
  • Wash slides in distilled water – 1 minute at room temperature.


  • This method should only be used if the chromogen substrate is alcohol insoluble.
  • Wash slides in 2 changes of 80% alcohol – 1 minute each at room temperature.
  • Wash slides in 2 changes of 95% alcohol – 1 minute each at room temperature.
  • Wash slides in 3 changes of 100% alcohol – 1 minute each at room temperature.
  • Wash slides in 3 changes of xylene – 1 minute each at room temperature.
  • Apply coverslip

LSBio's IHC-plus™ antibodies have been tested and identified as being optimal for use in immunohistochemistry (IHC) against formalin-fixed paraffin-embedded (FFPE) human tissues under LSBio's standardized IHC-plus™ protocol.  Each antibody is tested at multiple concentrations on more than 20 normal human tissue types, and when appropriate, multiple normal brain regions and/or cancer types.  LSBio's IHC protocol has been developed over the past 15 years as the most optimal method of immunolabeling FFPE tissues, the most common fixation method used by pathology labs worldwide.  A LifeSpan pathologist, with extensive experience evaluating IHC, analyzes the localization profile of each antibody, identifying positive and negative cell types, signal strength, subcellular and extracellular staining, and staining artifacts.  This information is then compared with all published expression and localization data available for the protein.  This enables LSBio to evaluate how each antibody behaves in IHC, including its specificity to the target protein, its sensitivity of detection, and any non-specific staining characteristics that it may display.  In order to be selected as an IHC-plus™ brand antibody, antibodies must have a close correlation to the published literature, be high affinity, display minimal staining artifacts, and have a high signal-to-noise ratio, such that its specific staining is considerably higher than its level of nonspecific background staining. Only the best antibodies receive approval and are given the designation as LSBio's premier IHC-plus™ antibodies. 

Learn more about LSBio's Immunohistochemistry (IHC) Validation.

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