title: Optimal antigen retrieval for ethanol-fixed cytologic smears.
abstract: BACKGROUND
  Antigen retrieval, a crucial technique for immunostaining, is often carried out on formalin-fixed, paraffin-embedded (FFPE) tissue sections. The role of antigen retrieval in immunostaining of ethanol-fixed smears remains unclear. The authors evaluated the effects of 2 common antigen retrieval procedures, heat-induced antigen retrieval and protease-induced antigen retrieval, for immunostaining using a broad panel of antibodies.
  
  
  METHODS
  Papanicolaou-stained ethanol-fixed smears from 36 surgical specimens were immunostained with 43 antibodies. Three widely used heat-induced antigen retrieval solutions, namely, citrate buffer (pH 6.0 and pH 7.0) and ethylenediaminetetraacetic acid solution (pH 8.0) for heat-induced antigen retrieval, and pronase were used. The staining results were compared between the ethanol-fixed smears and the corresponding FFPE tissue sections.
  
  
  RESULTS
  Heat-induced antigen retrieval was essential for all the 9 antibodies examined against nuclear antigens, and for 7 of 26 antibodies against cytoplasmic and cell membrane antigens. Superior results were obtained using lower-pH heat-induced antigen retrieval solutions for ethanol-fixed smears than was the case for FFPE tissue sections; use of citrate buffer (pH 6.0) was optimal for most antibodies. For 17 antibodies against cytoplasmic/cell membrane antigens, satisfactory results were obtained even without antigen retrieval on the ethanol-fixed smears, whereas antigen retrieval was necessary for detection on the FFPE tissue sections. Protease-induced antigen retrieval frequently exerted deleterious effects on ethanol-fixed smears. Despite antigen retrieval, detection of 2 lymphocytic markers failed on ethanol-fixed smears. This limitation was overcome by heat-induced antigen retrieval on formalin vapor-fixed smears.
  
  
  CONCLUSIONS
  In ethanol-fixed smears, most of the antibodies can be immunostained successfully without antigen retrieval treatment or mild heat-induced antigen retrieval using citrate buffer (pH 6.0). The optimal antigen retrieval condition for each antibody must be individually determined.
authors.affiliation: Division of Diagnostic Pathology and Cytology, Higashiyamato Hospital, Nangai, Higashiyamato, Tokyo, Japan. t.denda@yamatokai.or.jp
authors.forename: Tamami
authors.initials: T
authors.lastname: Denda
authors.forename: Shingo
authors.initials: S
authors.lastname: Kamoshida
authors.forename: Jumpei
authors.initials: J
authors.lastname: Kawamura
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Harada
authors.forename: Kenji
authors.initials: K
authors.lastname: Kawai
authors.forename: Sadahito
authors.initials: S
authors.lastname: Kuwao
mesh_terms: D000941:Antigens / Q000032:analysis / Q000276:immunology*; D003581:Cytodiagnosis / Q000379:methods; D000431:Ethanol / Q000494:pharmacology*; D005260:Female; D005404:Fixatives; D006358:Hot Temperature; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D009369:Neoplasms / Q000276:immunology* / Q000473:pathology* / Q000601:surgery; D016612:Paraffin Embedding; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D016707:Tissue Fixation / Q000379:methods
chemical_list: D000941:Antigens; D005404:Fixatives; D000431:Ethanol