Antigen Retrieval and Signal Amplification Protocol

AR originated as a method for improving immunoreactivity during IHC through trypsin digestion. The use of trypsin and other proteolytic enzymes, including proteinase K and Pepsin, for AR is now aptly termed protease-induced epitope retrieval (PIER). Efficacy of PIER is largely dependent on the target epitope, and optimization of incubation time and temperature, enzyme concentration, and the length of tissue fixation. PIER is not suitable for all epitopes and may damage tissue or epitope structure when performed incorrectly.

In 1991, Shi et al. described AR involving the submersion of FFPE tissues in boiling water, a process now known as heat-induced epitope retrieval, or HIER. HIER is generally superior in performance to PIER, with the exception of epitopes that may be damaged during the heating process and thus on which HIER would be ineffective. For optimal results, heating should be as even as possible, with at least 15–20 min of a constant boil recommended for HIER. Lower temperatures may also be used with extended incubation times. Several different buffers may be used for HIER; citrate buffer at pH 6.0, EDTA buffer at pH 8.0, and Tris-EDTA buffer at pH 9.0 are most commonly employed. Buffer pH can have a dramatic effect on HIER efficacy and experiments should be performed to determine the optimal pH for AR of any specific epitope. Experiments have shown both citrate and EDTA buffers to be effective for HIER; however, EDTA-based buffer systems are considered harsher than citrate buffers and may destroy more sensitive antigens and tissue structure.

Antigen retrieval alone may not be sufficient for generating a strong signal-to-background ratio when detecting low abundance proteins. Amplification of chromogenic precipitation of fluorescent detection greatly improves protein visualization in such cases. Secondary antibody conjugates are commonly employed for amplification during IHC. Binding between avidin and Biotin enables one such amplification method. Sections are incubated against biotinylated secondary antibodies raised against the primary antibody species. An avidin-biotinylated enzyme complex (ABC) solution is then introduced to the section. The ABC molecules then bind to both the biotinylated secondary antibody and to each other via the biotinylated enzyme (such as horseradish peroxidase), improving detection of the antigen. Importantly, endogenous enzyme activity must be blocked when performing ABC-mediated amplification; 3% hydrogen peroxide in methanol is sufficient to block endogenous peroxidase activity. Streptavidin may also be used for amplification in place of avidin, offering increased sensitivity by reducing nonspecific avidin binding.

Tyramide signal amplification (TSA) employs enzymatic amplification in a process similar to ABC, but with improved sensitivity. HRP catalyzes the deposition of fluorophore-conjugated tyramine at the site of the primary and secondary antibody complex. TSA also enables the simultaneous detection of multiple antigens in multiplex IHC with a high degree of sensitivity and specificity. Covalent bonds between the tyramine conjugate and proteins at the antigen site enables primary and secondary antibodies to be "stripped" from the sample without compromising the detection of the target protein so that additional targets can be labelled using primary antibodies from the same host species. TSA greatly increases the sensitivity of IHC, and primary antibody concentrations can be reduced at least twofold in TSA systems, highlighting its immense utility in detecting low-abundance proteins. Ultimately, amplification approaches such as ABC and TSA are most effective when combined with antigen retrieval, and together these approaches enable specific, high resolution protein detection via IHC. Protocols for both heat and protease-induced antigen retrieval, as well as for avidin– biotin complex based amplification and tyramide signal amplification are described herein.

Methods of Antigen Retrieval and Signal Amplification

Slide Preprocessing

1. Soften paraffin sections in the oven at 57–60 C for 40 min.
2. Dewax slides in xylene three times for 30 min each.
3. Rehydrate tissue in graded ethanol washes (100%, 90%, 70%, water) two times in each wash for 3 min per wash.
4. Quench endogenous peroxidase activity in 30% hydrogen peroxide in methanol for 20 min (20 mL 30% hydrogen peroxide +80 mL methanol).
5. Rinse and wash slides three times in PBS.
6. Proceed with antigen retrieval or amplification (protocols below).

Antigen Retrieval

Heat-Induced Epitope Retrieval (HIER)

1. Several different buffers can be used for HIER.
2. Preheat buffer to 85 C, using enough volume to ensure the sample on the slide is fully covered once submersed (see Note 5).
3. Place slides in the preheated buffer and incubate at 95–100 C for 15–30 min (see Note 6).
4. Remove slides form heat source and cool at room temperature for 20 min.
5. Rinse and wash slides three times in PBS.
6. Proceed with blocking, primary antibody labelling, and detection. If using ABC- or TSA-mediated amplification.

Protease-Induced Epitope Retrieval (See Note 7)

1. Several different proteases may be used for PIER.
2. Trypsin digestion.
   a) Preheat trypsin working solution to 37 C.
   b) Incubate sections with trypsin solution for 10–20 min at 37 C in a humidified chamber. Use enough volume to completely cover the sample (typically 100–300μL).
   c) Allow sections to cool at room temperature for 10 min.
   d) Proceed with step 5.
3. Pepsin digestion.
   a) Incubate sections with 100–300μL of Pepsin solution for 10–20 min at 37 C in a humidified chamber.
   b) Allow sections to cool at room temperature for 10 min.
   c) Rinse and wash sections two times with PBS-Tween.
   d) Proceed with step 6.
4. Proteinase K digestion.
   a) Incubate sections with 100–300μL of Proteinase K solution for 2–4 min at room temperature.
   b) Proceed with step 5.
5. Rinse and wash slides three times in PBS.
6. Proceed with blocking, primary antibody labelling, and detection. If using ABC- or TSA-mediated amplification.

Amplification (See Note 8)

Amplification Using Biotinylated Secondary Antibodies and ABC

1. Block sections in the appropriate blocking buffer for 2 h at room temperature.
2. Tap off blocking buffer and incubate slides in primary antibody diluted in 0.1% PBS-BSA.
3. Rinse and wash slides three times in PBS.
4. Incubate slides in biotinylated secondary antibody diluted 1: 200 in 0.1% PBS-BSA for 1 h at room temperature.
5. Prepare ABC (see Note 9). Add 2 drops of reagent A solution and 2 drops of reagent B solution to 5 mL of 0.1% PBS-BSA. Allow the ABC solution to sit for 1 h before use.
6. Rinse and wash slides three times in 1XPBS.
7. Apply ABC solution for 1 h at room temperature.
8. Rinse and wash slides three times in 1XPBS.
9. Visualize proteins using DAB. Apply DAB solution for 3 min or less.
10. Rinse and wash slides three times with tap water.
11. Perform a light nuclear counterstain, rinse and wash slides in tap water between each step.
    a) Place slides in hematoxylin for 4–5 min.
    b) Remove excess hematoxylin with one quick dip in acid alcohol.
    c) Reblue slides in ammonia water.
12. Dehydrate slides in graded ethanol washes (70%, 85%, 100%), two times in each wash for 3 min each.
13. Clear slides in xylene for 30 min.
14. Mount using Permount.

Tyramide Signal Amplification

1. Apply PerkinElmer TNB blocking buffer to sections for 30 min at room temperature.
2. Dilute the primary antibody in TNB blocking buffer. Tap off blocking buffer and apply the diluted primary antibody solution to the sections. Incubate at least 1 h (see Note 10).
3. Rinse and wash slides three times in PerkinElmer TNT wash buffer.
4. For HRP-conjugated secondary antibodies, dilute in TNB blocking buffer and incubate for 1 h at room temperature.
5. For biotinylated secondary antibodies, carry out the following.
   a) Dilute the secondary antibody in TNB blocking buffer.
   b) Incubate for 1 h at room temperature.
   c) Rinse and wash slides three times in TNT wash buffer.
   d) Incubate slides with streptavidin–HRP conjugate diluted in TNB blocking buffer for 30 min at room temperature.
6. Rinse and wash slides three times in TNT wash buffer.
7. For chromogenic detection:
   a) Dilute reconstituted Fluorescein Tyramide 1:50 in PerkinElmer Amplification diluent. Incubate slides in Fluorescein Tyramide solution for 10 min at room temperature.
   b) Rinse and wash slides three times in TNT wash buffer.
   c) Incubate slides in anti-fluorescein-HRP (for chromogenic detection with DAB) or anti-fluorescein-AP (for chromo-genic detection with LPR) diluted 1:100 in TNB blocking buffer for 30 min at room temperature.
   d) Rinse and wash slides three times in TNT wash buffer.
   e) Incubate slides in DAB (for HRP conjugate) for 3 min at room temperature or LPR (for AP conjugate) for 10 min at room temperature.
   f) Rinse and wash slides three times in tap water (for DAB) or PBS (for LPR).
   g) Perform a light hematoxylin counterstain.
   h) Dehydrate sections, clear in xylene, and mount using Permount.
8. For fluorescent detection:
   a) Dilute reconstituted Fluorophore Tyramide 1:50 in PerkinElmer Amplification diluent.
   b) Incubate slides in Fluorophore Tyramide solution for 10 min at room temperature.
   c) Rinse and wash slides three times in TNT wash buffer.
   d) Perform a nuclear counterstain with DAPI diluted to a concentration of 1μg/mL PBS. Alternatively, mounting media containing DAPI may be used in place of a separate counterstain step.
   e) Rinse and wash slides in TBST then distilled water for 2 min each.
   f) Mount with a fluorescence-safe aqueous mounting medium.