Immunohistochemistry Protocol for Beta Amyloid Products using USA Detection Kit

 

Introduction

 

Protocol can be used for Beta Amyloid products that list “IHC” as an application on the datasheet (e.g. clones 4G8, 6E10, etc).

Use with Ultra Streptavidin Detection Kit (BioLegend Cat #929501/SIG-32250). All steps should be done in a humidity chamber such as BioLegend Cat #926301/SIG-31031.

 

Protocol Steps

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1. Clear Slides: Remove paraffin and hydrate the tissue
   Note: If using frozen sections, allow slides to come to room temperature for 15 minutes & proceed to step (F) only
    
   • A. Xylene - 5 minutes in each of (3) different 250 mL containers
   • B. 100% alcohol - 5 minutes in each of (3) different 250 mL containers
   • C. 95% alcohol - 3 minutes in (1) 250 mL container
   • D. 70% alcohol - 3 minutes in (1) 250 mL container
   • E. Water -1 minutes in each of (3) different 250 mL containers
   • F. H₂O₂ (3%) - 15 minutes in (1) 250 mL container


2. Rinse slides with lab grade water.
   Note: Lab grade filtered water such as injection grade, cell culture grade, Reverse Osmosis De-Ionization (RODI).  


3. Antigen Retrieval (refer to product datasheet, not always required)
    
   1. 70% Formic Acid - incubate the slides for 20 minutes at room temperature.
      Note: This antigen retrieval step is harsh on the tissue. If using frozen sections reduce time to 5-10 minutes or omit if tissue falls off the slide.
   2. Rinse Slides with 1X PBS.


4. Apply serum block for at least 5 minutes. Do not wash after this step.


5. Blot off serum block.  


6. Apply primary antibody - dilute to 1 µg/mL in PBS.  


7. Incubate primary antibody 60 minutes at room temperature.


8. Rinse slides with 1X PBS.


9. Apply USA Linking reagent - 20 minutes incubation.


10. Rinse slides with 1X PBS.  


11. Apply Labeling Reagent - 20 minutes incubation.


12. Rinse with 1X PBS.  


13. Apply chromogen - 5 minutes incubation. Dilute according to manufacturer's instructions.
     
    1. AEC Chromogen: 20 µL AEC chromogen + 1 mL AEC substrate buffer.
    2. DAB Chromogen: 40 µL DAB chromogen + 1 mL DAB substrate buffer.


14. Rinse slides with lab grade water.  


15. Counterstain
     
    1. Submerge slides in Mayer’s Hematoxylin for 30 seconds.
    2. Rinse under running lab grade water for 1 minute or until water is clear.
    3. Submerge slides in Bluing Reagent for 1 minute.
    4. Rinse under running lab grade water for 1 minute.


16. Clear slides: Dehydrate the tissue.
     
    1. 95% alcohol 3 minutes in (1) 250 mL container.
    2. 100% alcohol 5 minutes in each of (3) different 250 mL container.
    3. Xylene 5 minutes in each of (3) different 250 mL container.


17. Cover slip slide using Permanent Aqueous Mounting Medium.
    Note: Do not use xylene based mount with AEC Chromogen as it will dissolve the chromogen.  

 

General Tips & FAQ

FAQ:

• Do I need to perform antigen retrieval on my formalin-fixed, paraffin-embedded samples prior to staining?
  • In most cases, this is true. Antigen retrieval helps both the accessibility of the antibody to the tissue and also counteracts the fixation effects on the recognized epitopes. Check the application references for any additional details for IHC or IF experiments.
• Can antibody X be used for immunohistochemistry? What concentration do I use?
  • Typical concentrations of monoclonal antibodies for use in IHC are from 5-25 µg/ml. Polyclonal antibodies can be used at a range of 1-10 µg/ml. We will indicate on the datasheet if an antibody has been published for use in this application. In addition, you can do a literature search with the clone name and immunohistochemistry/paraffin/frozen to see what the protocol details are.
• Why should I block endogenous peroxidase activity? Do I use PBS or methanol to make the H₂O₂ solution?
  • Endogenous peroxidases will react with the substrate solution (hydrogen peroxide and chromogen, e.g. DAB), leading to false positives. This non-specific background can be significantly reduced by pre-treatment of the sample with hydrogen peroxide before incubation with HRP-conjugated antibody.
    
    Morphology of blood smears and peroxidase-rich tissues can sometimes be damaged by the hydrogen peroxide. Diluting the hydrogen peroxide in methanol is the best choice for fragile samples where preservation of morphology is required.
    
    However, some cell surface protein markers are very sensitive to methanol or hydrogen peroxide quenching, reducing the staining of antigenic site, particularly on frozen sections. Using hydrogen peroxide in PBS is suggested for cell surface or membrane proteins. Another protocol modification is to quench with peroxide after first incubating section or cells with the primary antibody.