Griffin:Antigen Retrieval Technique

• Korey Griffin Resource List

Antigen Retrieval optimizes tissue antigen detection of tissue sections. Pretreatment with an antigen retrieval reagent create a greater exposure for antibodies to bind antigenic sites. Antigen Retrieval methods are not equally applicable to all antibodies.

High temperature (>95C) treatment

Heat treatment >95°C in certain buffer solutions can reconstitute protein antigenicity that would otherwise be nonreactive due to formaldehyde fixation and/or paraffin embedding. Heat treatment can disrupt formalin dependent aldehyde cross-linking of proteins, and restore antigenic structure.

A standard Rice cooker is an effective heat/steam source for antigen unmasking

10-20 minute heat treatment is a common range that is widely effective. The cooling process taking ~20 minutes. The main mechanism of heat-induced antigen retrieval is disruption of the cross-links and pH is also an essential factor for proper refolding of epitopes. For this reason a low pH and high pH buffer is a good contrast in optimizing the staining protocol.

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.

Below are 3 options to consider;

10 mM sodium citrate buffer, pH 6.0

Summary: Heat at 95º C for 5 minutes. Top off with fresh buffer and heat at 95º C for 5 minutes (optimal incubation time may vary for each tissue type). Allow slides to cool in the buffer for approximately 20 minutes. Wash in deionized H2O three times for 2 minutes each. Aspirate excess liquid from slides.

Procedure:

Materials: Citrate or Glycine-EDTA buffer, a microwaveable plastic container with slide holder and a microwave with a rotating dish.

I) For consistency, fill the slide holder with slides even if only a couple of slides with tissues are being stained. Use clean slides with no tissue for that.

II) Fill the container with the citrate buffer, which is kept at ambient temperature.

III) Place the container with the slides and the buffer in the microwave and bring the buffer to a boil with the microwave at full power. This may take around 2 minutes depending on the microwave used. As soon as the buffer begins to boil the power is reduced to about 30% to allow intermitent boiling of the buffer. Keep the slides for 7 minutes under these conditions. This is done so the tissues don't come off the slides due to continuous boiling. Note: the amount of power used for this step will need to be determined for each microwave.

IV) The container is left at ambient temperature to cool down.

V) Once the buffer is near ambient temperature the slides with tissues are removed, washed briefly with 1xPBS and we proceed with the peroxidase blocking and antibody staining.

50 mM glycine-HCl buffer, pH 3.5, with 0.01% (w/v) EDTA

Summary: Heat at 95º C for 5 minutes. Top off with fresh buffer and heat at 95º C for 5 minutes (optimal incubation time may vary for each tissue type). Allow slides to cool in the buffer for approximately 20 minutes. Wash in deionized H2O three times for 2 minutes each. Aspirate excess liquid from slides.

Procedure:

Materials: Citrate or Glycine-EDTA buffer, a microwaveable plastic container with slide holder and a microwave with a rotating dish.

I) For consistency, fill the slide holder with slides even if only a couple of slides with tissues are being stained. Use clean slides with no tissue for that.

II) Fill the container with the citrate buffer, which is kept at ambient temperature.

III) Place the container with the slides and the buffer in the microwave and bring the buffer to a boil with the microwave at full power. This may take around 2 minutes depending on the microwave used. As soon as the buffer begins to boil the power is reduced to about 30% to allow intermitent boiling of the buffer. Keep the slides for 7 minutes under these conditions. This is done so the tissues don't come off the slides due to continuous boiling. Note: the amount of power used for this step will need to be determined for each microwave.

IV) The container is left at ambient temperature to cool down.

V) Once the buffer is near ambient temperature the slides with tissues are removed, washed briefly with 1xPBS and we proceed with the peroxidase blocking and antibody staining.

0.05% citraconic anhydride solution, pH 7.4

Formaldehyde reacts predominantly with the lysyl residues and forms intramolecular cross-links. Citraconic anhydride reacts with the free amino groups of proteins and replaces the positively charged NH3 groups of lysyl residues with negatively charged carboxyl groups. Citraconic anhydride solution efficiently releases formaldehyde cross-links. Heat deparaffinized formalin fixed tissue sections in 0.05% citraconic anhydride solution, pH 7.4, at 98C for 45 min., followed by 3 wash steps.

1. Yamashita S and Okada Y. Mechanisms of heat-induced antigen retrieval: analyses in vitro employing SDS-PAGE and immunohistochemistry. J Histochem Cytochem. 2005 Jan;53(1):13-21. DOI:10.1177/002215540505300103 |
2. Namimatsu S, Ghazizadeh M, and Sugisaki Y. Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method. J Histochem Cytochem. 2005 Jan;53(1):3-11. DOI:10.1177/002215540505300102 |

Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0)

Tris-EDTA based solution utilizes heat to dissociate covalent cross-links.

• Tris Base  : 1.21 g
• EDTA  : 0.37 g
• Distilled water  : 1000 ml (100 ml to make 10x, 50 ml to make 20x)

Mix to dissolve. pH 9.0, add 0.5 ml of Tween 20, mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.

Procedure:

• 1. Deparaffinize sections in 2 changes of xylene, 5 minutes each. 

• 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water.
• 3. Pre-heat steamer or water bath with staining dish containing Tris-EDTA Buffer until temperature reaches 95-100 °C.
• 4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user).
• 5. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes.
• 6. Rinse sections in PBS Tween 20 for 2x2 min.
• 7. Block sections with for 30 minutes.
• 8. Perform avidin/biotin blocking if necessary.
• 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
• 10. Rinse sections with PBS Tween 20 for 2x2 min.
• 11. Block sections with peroxidase blocking solution for 10 minutes.
• 12. Rinse with PBS Tween 20 for 3x2 min.
• 13. Proceed to standard immunohistochemistry protocol.

Low-temperature (<80C) treatment

Sodium Citrate Buffer 80C bath

• Low-temperature antigen retrieval (LTAR): 80C, 10mM Sodium Citrate, 2 hours w/ trypsin pretreatment

Enzymatic Treatment

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.

Trypsin

• Incubate sections for 10–20 minutes in 0.1% Trypsin PBS at room temperature. Wash slides several times in deionized H2O. Aspirate excess liquid from slides.

Pepsin

• Incubate sections for 10–20 minutes in 0.1% pepsin in 0.01 N HCl at room temperature. Wash slides several times in deionized H2O. Aspirate excess liquid from slides.

Detergent Treatment

• Saponin interacts with membrane cholesterols, forming pores and/or removing cholesterols from the membrane
• Incubate sections for 30 minutes in 0.05% saponin in deionized H2O at room temperature. Wash at least three times in PBS. Aspirate excess liquid from slides.
• Saponin is favorable for intracellular membrane antigens in many organelles.

Reference

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