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Alcohol based tissue fixation as an alternative for formaldehyde: influence on immunohistochemistry
  1. Hendrik F van Essen,
  2. Marina A M Verdaasdonk,
  3. Sabrina M Elshof,
  4. Roel A de Weger,
  5. Paul J van Diest
  1. Department of Pathology, University Medical Center Utrecht, The Netherlands
  1. Correspondence to Professor Paul J van Diest, Department of Pathology, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands; p.j.vandiest{at}


Background Formaldehyde is commonly used in histopathology to fix tissue. Not only are the carcinogenic properties of this solution a hazard to the people in the workplace, it is also a major burden on the environment, and it crosslinks molecular groups that hinder immunohistochemistry.

Aims The influence of two new alcohol based non-crosslinking fixatives on immunohistochemical staining properties was tested on various tissues.

Methods Fresh tissue samples were cut into three equal pieces, which were then fixed in the alcohol based fixatives Boonfix or RCL2, or in neutral buffered formaldehyde (NBF) as control. After fixation, tissue was routinely processed to paraffin sections. Deparaffinised slides were blocked for endogenous peroxidase and subsequently submitted to the usual NBF based immunohistochemical protocols for 85 different common antibodies either not requiring antigen retrieval (AR), or AR in citrate buffer, EDTA or pepsin.

Results NBF fixed tissues provided significantly better immunostaining results (84% good staining) than RCL2 (66% good) and Boonfix (60%). The lesser performance of RCL2 and Boonfix was especially caused by pepsin AR which caused significant tissue damage. Omission of pepsin AR resulted in better immunostaining for these antibodies for RCL2 fixed tissues which were overall no longer significantly worse than NBF fixed tissue.

Conclusion Tissues fixed in non-crosslinking alcohol based fixatives can successfully be immunohistochemically stained for most antibodies following the usual NBF based protocols. Omission of pepsin pretreatment seems to be important to retain proper morphology of immunostained tissues preserved in alcohol based fixatives. Therefore, when switching to less toxic and non-carcinogenic alcohol-based fixatives like RCL2, no major changes in the daily routine of immunohistochemistry are anticipated.

  • Formaldehyde
  • fixation
  • histopathology
  • immunohistochemistry

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  • Competing interests None.

  • Provenance and peer review Not commissioned; externally peer reviewed.