Antigen Retrieval Method for Free Floating Sections




Description: We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating sections. We found that the best results were obtained with a 30 min incubation in a 10–50 mM sodium citrate solution (pH 8.5–9.0) preheated to and maintained at 80°C in a water-bath, followed by 30 min incubation in 0.3–3% nonfat dry milk to reduce nonspecfic staining. This method is highly effective for both 40 um free floating sections, slide-mounted cryostat sections and paraffin-embedded slide-mounted sections.


Solutions and Reagents:  


Sodium Citrate Buffer (10mM Sodium Citrate Buffer, pH 8.5):

      Tri-sodium citrate ---------------------- 2.94 g

      Distilled water -------------------------- 1000 ml

      Mix to dissolve. Adjust pH to 8.5 and store this solution at room temperature for 3 months or at 4 C for longer storage.



  1. Rinse sections three times for 5 min each in 0.1 M PB (pH 7.4).

  2. Transfer the sections to 10 mM sodium citrate buffer (pH 8.5) preheated to 80°C in water bath.

  3. Maintain this temperature and keep sections in this solution for 30 min.

  4. Retain the sections in this solution while allowing the solution to cool to room temperature.

  5. Rinse the sections three times for 5 min each in 0.1 M PB (pH 7.4).

  6. Immerse the sections in 2% nonfat dry milk in 0.1 M PB (pH 7.4) containing 0.3% Triton X-100–0.01% sodium azide for 30–60 min.

  7. Incubate in the primary antibody and complete immunohistochemical staining steps as desired.





1. Yun Jiao, et al (1999) A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections. Journal of Neuroscience Methods 93:149–162.


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