Correspondence

More on p53 Antigen Loss in Stored Paraffin Slides

N Engl J Med 1995; 333:1507-1508November 30, 1995

Article

To the Editor:

Prioleau and Schnitt (June 1 issue)1 reported a potential problem of p53 antigen loss in slides that had been stored at room temperature for two months. We examined slides that had been cut from formalin-fixed, paraffin-embedded blocks containing a p53-positive colorectal carcinoma and accidentally stored at room temperature for periods of 6 to 14 months. We found that the p53 reactivity could be restored with a method of antigen retrieval2 based on heating the slides in a microwave oven.

We examined slides from four patients with colorectal carcinoma in situ that did not stain for p53 by conventional methods after long-term storage, despite initial positive findings. We soaked the slides in 10 mM sodium phosphate buffer (pH 6.8) and heated them three times in a household microwave oven at 500 W for five minutes, with a one-minute pause between heatings. we then stained the slides for p53 with an anti-p53 monoclonal antibody (PAb 1801, Oncogene Science, manhasset, N.Y.) and a Vectastain ABC kit (Vector Laboratories, Burlingame, Calif.). In all cases, p53 antigen was detected and the staining was excellent. The results were consistent with those of the initial tests.

Heating slides in a microwave oven may be an effective method of antigen retrieval before immunohistochemical staining for p53, even when the slides have been stored at room temperature for long periods.

Junji Kato, M.D., Ph.D.
Sumio Sakamaki, M.D., Ph.D.
Yoshiro Niitsu, M.D., Ph.D.
Sapporo Medical University, 060 Sapporo, Japan

2 References

1. 1

   Prioleau J, Schnitt SJ. p53 Antigen loss in stored paraffin slides. N Engl J Med 1995;332:1521-1522
   Full Text | Web of Science | Medline

2. 2

   Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991;39:741-748
   CrossRef | Web of Science | Medline

Author/Editor Response

The authors reply:

To the Editor: Previous studies have clearly shown that using a microwave oven for antigen retrieval is necessary for optimal p53 staining.1,2 In our study of mammary ductal carcinoma in situ, both stored and unstored slides were subjected to microwave heating in citrate buffer (pH 6) for two five-minute periods before p53 immunostaining. We found that the intensity of staining for p53 in microwave-treated stored slides was consistently less than that in unstored slides so treated. In contrast, Kato et al. studied p53 staining in a different type of tumor (colorectal carcinoma), used a different method of microwave heating, and apparently subjected stored slides to microwave treatment, but not unstored slides. Although the effects of the microwave heating of stored slides appear to differ in these two studies, methodologic differences make direct comparison of the results impossible.

Although slides clearly must be heated in a microwave oven for optimal results of immunohistochemical staining for p53, our experience has been that even with such treatment, stored slides did not stain for p53 as well as unstored slides. It is possible that other methods of antigen retrieval (which may or may not include microwave heating) may improve the results of p53 staining in stored slides.

John E. Prioleau, B.A.
Stuart J. Schnitt, M.D.
Beth Israel Hospital, Boston, MA 02215

2 References

1. 1

   Linden MD, Nathanson SD, Zarbo RJ. Evaluation of anti-p53 antibody staining: quality control and technical considerations. Appl Immunohistochem 1994;2:218-224
   

2. 2

   Resnick JM, Cherwitz D, Knapp D, Uhlman D, Niehans GA. A microwave method that enhances detection of aberrant p53 expression in formalin-fixed, paraffin-embedded tissues. Arch Pathol Lab Med 1995;119:360-366
   Web of Science | Medline

Citing Articles (5)

Citing Articles

1. 1

   Clive R. Taylor, Shan-Rong Shi, Nancy J. Barr. 2011. Techniques of Immunohistochemistry. , 1-41.
   CrossRef

2. 2

   E. Evke, F. Z. Minbay, S. G. Temel, Z. Kahveci. (2009) Immunohistochemical detection of p53 protein in basal cell skin cancer after microwave-assisted antigen retrieval. Journal of Molecular Histology 40:1, 13-21
   CrossRef

3. 3

   Martina Mirlacher, Marlis Kasper, Martina Storz, Yvonne Knecht, Ursula Dürmüller, Ronald Simon, Michael J Mihatsch, Guido Sauter. (2004) Influence of slide aging on results of translational research studies using immunohistochemistry. Modern Pathology 17:11, 1414-1420
   CrossRef

4. 4

   Lambert J. C.M. van den Broek, Marc J. van de Vijver. (2000) Assessment of Problems in Diagnostic and Research Immunohistochemistry Associated With Epitope Instability in Stored Paraffin Sections. Applied Immunohistochemistry & Molecular Morphology 8:4, 316-321
   CrossRef

5. 5

   U. MANNE, R. B. MYERS, S. SRIVASTAVA, W. E. GRIZZLE. (1997) Re: Loss of Tumor Marker-Immunostaining Intensity on Stored Paraffin Slides of Breast Cancer. JNCI Journal of the National Cancer Institute 89:8, 585-586
   CrossRef