Description: Although AR by heating is widely effective, the majority ofthe existing methods are designed for paraffin-embedded sections. Frozen sections have been generally exempt from these methods because such sections are fragile and are easily destroyed by heating. A recent report demonstrates a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases.

Solutions and Reagents:  

Citrate Buffer (10mM Sodium Citrate Buffer, 0.05% Tween 20, pH 6.0):

• Tri-sodium citrate ---------------------- 2.94 g
• Distilled water -------------------------- 1000 ml

 Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage.

Procedure:

1. Prepare tissues fixed with 4% paraformaldehyde in 0.1M PB, pH 7.5. The tissue blocks should be cut toa proper size (e.g., slices 3–5 mm thick).
2. Immerse the tissue blocks in a retrieval solution (10 mM sodium citrate buffer, pH 6.0) at 4 ºC overnight.
3. Place the tissue blocks in a small, heat-resistant basket and immerse in boiling retrieval solution (200–500 ml) with gentle stirring using a hot plate for 3–5 min. For heating, a conventional burner can also be used.
4. Immediately place the tissue blocks in cold30% sucrose in PBS and incubate at 4 ºC until the blocks sink.
5. Immerse the tissue blocks in an embedding medium and freeze quickly with crushed dry ice. The frozen tissue blocks can now be stored at -80 ºC.
6. Cut frozen sections with a cryostat and mount them on glass slides. Free-floating sections can also be used.
7. Dry the sections well for more than 1 day or several days at RT to prevent detachment of the sections during processing.
8. Proceed to standard immunohistochemistry procedure.

References:

1. Ino H (2003) Antigen retrieval by heating en bloc for pre-fixed frozen material. J Histochem Cytochem. 51(8):995-1003. PubMed Abstract

2. Evers P, Uylings HB (1994) Effects of microwave pretreatment on immunocytochemical staining of vibratome sections and tissue blocks of human cerebral cortex stored in formaldehyde fixative for long periods. J Neurosci Methods. 55(2):163-72. PubMed Abstract