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Technical Tips

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Technical Tips

This page contains frequently asked questions and answers involving key steps for immunohistochemical staining and related topics.

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Antigen Retrieval Technique

 

What is antigen retrieval technique

Which antigen retrieval method should I use for a specific antibody

How to set up a test for a new antibody with various antigen retrieval methods

I have tested my antibody with various antigen retrieval methods, but still no results

In heat induced epitope retrieval (HIER) methods, which one is the best

What are key factors to be considered when I do pretreatment with HIER

In proteolytic enzyme induced epitope retrieval (PIER) methods, which one is the best

What are key factors to be considered when I do pretreatment with PIER

Do I need to do antigen retrieval for frozen sections

 

Blocking Endogenous Peroxidase Activity

 

Why should I block endogenous peroxidase activity

How do I know if my tissues contain endogenous peroxidase activity

What chemicals should I use  to block endogenous peroxidase activity

What should I know when handling hydrogen peroxide

What solutions or reagents should I use to dilute hydrogen peroxide

What concentration of hydrogen peroxide is commonly used

Where should I do hydrogen peroxide blocking during IHC procedure

For how long the sections should be incubated in blocking solution

I blocked with hydrogen peroxide, but still getting background staining

How to block endogenous peroxidase activity on frozen sections

What are alternative methods for blocking endogenous peroxidase activity

 

Blocking Endogenous Biotin

 

Why should I block endogenous biotin

How do I know if my tissues contain endogenous biotin

What chemicals/reagents to use for blocking endogenous biotin

Where should I block endogenous biotin during IHC procedure

What is the procedure for blocking endogenous biotin

Why do I need two steps to block endogenous biotin

I blocked with avidin/biotin blocking reagents, but still getting background staining

 

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What is antigen retrieval technique

Click here for the detailed description about antigen retrieval techniques. Back to top

 

Which antigen retrieval method should I use for a specific antibody

Some companies test their antibodies for immunohistochemistry application and will include a suggested antigen retrieval method in the datasheet (note: some of the suggested method is not optimal). if no one of above gives you a clear answer, a test is needed. Back to top

 

How to set up a test for a new antibody with various antigen retrieval methods

Initially, you may want to test two methods from HIER such as citrate buffer (pH 6) and Tris-EDTA (pH 9), and one or two methods from PIER such as trypsin and/or pronase. This should give you a good chance to obtain an optimal one for a specific antibody. Back to top

 

I have tested my antibody with various antigen retrieval methods, but still no results

The antibody may not be appropriate for formalin-fixed, paraffin-embedded tissues (some antibodies only work for frozen sections) or the antibody itself has a problem and you may need to switch to a different antibody. Back to top

 

In heat induced epitope retrieval (HIER) methods, which one is the best

Citrate buffer (pH 6) method is one of the commonly used. Some studies showed that Tris-EDTA (pH 9) or Tris-HCl (pH 10) is superior. A test is probably the best way to know which one is the best for a specific antibody. Back to top

 

What are key factors to be considered when I do pretreatment with HIER

(1) Temperature of retrieval solution should be around 95 C; (2) Incubation time should be at least 10 minutes and it is usually around 20 minutes; (3) pH value of retrieval solution is depending on which solution you are using. Back to top

 

In proteolytic enzyme induced epitope retrieval (PIER) methods, which one is the best

Trypsin method seems to be the commonly used one. The concentration, incubation time and temperature probably are more important factors to consider. Again, a test is the best way to know which one is the best for a specific antibody. Back to top

 

What are key factors to be considered when I do pretreatment with PIER

(1) Concentration of enzyme is usually 0.05-0.1% depending on type of tissue and fixation; (2) Incubation time could be 5-30 minutes and 10-15 minutes is commonly used; (3) Incubation temperature is usually at 37 C. Back to top

 

Do I need to do antigen retrieval for frozen sections

Antigen retrieval is not needed for immunostaining of fresh frozen sections. Because antigen retrieval is to break the cross-links formed by formalin fixation and fresh frozen sections are sectioned and fixed with ice cold acetone for 5-10 minutes. Back to top

 

Why should I block endogenous peroxidase acitivity

Some cells or tissues contain endogenous peroxidase. Using HRP conjugated antibody may result in high, non-specific background staining. This non-specific background can be significantly reduced by pre-treatment of cells/tissues with hydrogen peroxide prior to incubation with HRP conjugated antibody. Back to top

 

How do I know if my tissues contain endogenous peroxidase activity

Incubate your tissue sections with DAB after rehydration to water. If it turned brown, the tissue contains endogenous peroxidase and a blocking step is needed. Back to top

 

What chemicals should I use to block endogenous peroxidase activity

Hydrogen peroxide (H2O2), a blocking agent in immunohistochemistry, is commonly used to block endogenous peroxidase acitivity. Pre-treatment with saturating amounts of hydrogen peroxide results in the irreversible inactivation of endogenous peroxidase. Back to top

 

What should I know when handling hydrogen peroxide

Hydrogen peroxide should be stored in the refrigerator and protected from sunlight in order to slow itís thermal decomposition. Never return unused hydrogen peroxide to the original container. Do not pipette from a reagent bottle of 30% or greater of hydrogen peroxide. If contaminated with certain metals or their salts, hydrogen peroxide can decompose violently. The bottle is vented because hydrogen peroxide decomposes in the presence of traces of impurities, yielding oxygen and water. This could cause dangerously high pressure in a tightly capped container. Back to top

 

What solutions or reagents should I use to dilute hydrogen peroxide

Methanol, PBS, distilled water or saline can be used to dilute hydrogen peroxide. Morphology of blood smears and peroxidase-rich tissues could be damaged by the aqueous hydrogen peroxide solution. Therefore, methanol is a better choice in this case. Some cell surface markers are very sensitive to methanol/hydrogen peroxide quenching, reducing the staining of antigenic site, particularly on frozen sections. So using hydrogen peroxide in PBS is recommended for cell surface or membrane markers. Back to top

 

What concentration of hydrogen peroxide is commonly used

3% hydrogen peroxide is commonly used to block endogenous peroxidase activity. However, certain tissues/cells/antigen (i.e. cell surface markers such as CD4) can be destroyed by high concentration of hydrogen peroxide. So a lower concentration (0.3%) should be used. Back to top

 

Where  should I do hydrogen peroxide blocking during IHC procedure

The blocking can be done (1) after rehydration to water and before antigen retrieval, (2) after antigen retrieval and before primary antibody incubation, (3) after primary antibody incubation, or (4) after biotinylated secondary antibody incubation. For certain antigens such as CD4 and CD8, hydrogen peroxide blocking has detrimental effect on the epitopes, thus reduce intensity of antibody staining. Therefore, blocking after primary antibody or secondary antibody incubation is recommended. Back to top

 

For how long the sections should be incubated in blocking solution

It can be 5 to 60 minutes depending on concentration of hydrogen peroxide, diluting reagent, tissue type, and fixation. 10-15 minutes incubation is commonly used for formalin-fixed, paraffin embedded tissue sections. Back to top

 

I blocked with hydrogen peroxide, but still getting background staining

The hydrogen peroxide may be expired. Try a fresh bottle of hydrogen peroxide and it may solve the problem. In addition, the tissue may contain endogenous biotin as well, so avidin/biotin blocking step may be needed. Finally, if the background problem still persists, you may consider switching to a different detection system, such as AP system or immunofluorescence method. Back to top

 

How to block endogenous peroxidase activity on frozen sections

Immerse slides in fresh made 0.3% hydrogen peroxide in 0.1% sodium azide for 10-15 minutes (to make the blocking solution, add 5ml of 3% hydrogen peroxide to 45 ml of 0.1% sodium azide and mix well). An alternative is to use 0.3% hydrogen peroxide in methanol for 20-30 minutes since methanol accelerates the destruction of the heme groups so a lower concentration of hydrogen peroxide can be used for longer incubation time.  Back to top

 

What are alternative methods for blocking endogenous peroxidase activity

One alternative is to incubate sections in 0.36% beta-glucose-0.01% glucose oxidase-0.013% sodium azide in PBS for 60 minutes at 37 C. Another method is to incubate sections in 0.01% periodic acid for 10 minutes followed by 0.01% sodium borohydride in water for 2 minutes. Back to top

 

Why should I block endogenous biotin

Some cells or tissues contain endogenous biotin. Using avidin-biotin method may result in high, non-specific background staining. This non-specific background can be significantly reduced by pre-treatment of cells/tissues with avidin/biotin blocking reagents prior to the incubation of biotinylated antibody . Back to top

 

How do I know if my tissues contain endogenous biotin

Kidney, liver and spleen usually contain high level of endogenous biotin and a blocking step is needed when you are using avidin-biotin system for these tissues. You can also do a simple test by incubating sections directly with ABC complex or streptavidin-HRP and then DAB, but be sure to apply hydrogen peroxide first to rule out the background staining caused by endogenous peroxidase. Back to top

 

What chemicals/reagents to use for blocking endogenous biotin

Two chemicals, avidin and biotin, are needed. You can purchase from Sigma and make 0.05% avidin and 0.005% biotin in PBS. Commercial avidin/biotin blocking kits are also available from many companies. Dr. Rodney Miller has also used egg white as a source of avidin and skim milk as a source of biotin to block endogenous biotin successfully,  but water should be used to rinse sections between steps since PBS will precipitate out proteins in the egg white. Back to top