title: Immunostaining of cell preparations: a comparative evaluation of common fixatives and protocols.
abstract: Immunostaining of cytologic preparations has been beset by problems of inconsistency, high background staining, and the requirement of different fixatives for different antigens. This study sought to identify a universal fixative and a simple fixation protocol suitable for a wide range of tissue antigens commonly employed for cytologic diagnosis. In an analysis of 23 fixation protocols involving acetone, acetone/methanol, acetone/formalin, glutaraldehyde, ethanol, methanol, and formal saline, fixation in 0.1% formal saline overnight at 27 degrees C followed by 10 min fixation in 100% ethanol produced the most consistent and optimal preservation of immunoreactivity which could be further enhanced by pre-treatment with microwaves for epitope retrieval. Blocking of endogenous peroxidase was not necessary with this fixation protocol. Provided the smears were well air-dried (for at least 14 hr) prior to immersion in formal saline, there was no need to employ adhesive-coated glass slides. The smears could be kept at 27 degrees C (room temperature) for at least 7 days and at -70 degrees C for 5 wk without loss of immunoreactivity as air-dried smears or after fixation in formal saline. One hundred percent acetone and 100% ethanol produced good morphology and immunoreactivity but a high level of background staining, whereas acetone-based mixtures resulted in inconsistent immunostaining.
authors.affiliation: Division of Tissue Pathology, Institute of Medical and Veterinary Science, Adelaide, South Australia.
authors.forename: C
authors.initials: C
authors.lastname: Suthipintawong
authors.forename: A S
authors.initials: AS
authors.lastname: Leong
authors.forename: S
authors.initials: S
authors.lastname: Vinyuvat
mesh_terms: D000096:Acetone / Q000737:chemistry; D000311:Adrenal Glands / Q000473:pathology; D000431:Ethanol / Q000737:chemistry; D005260:Female; D005404:Fixatives / Q000737:chemistry*; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D008099:Liver / Q000473:pathology; D008297:Male; D009215:Myometrium / Q000473:pathology; D010525:Peripheral Nerves / Q000473:pathology; D011467:Prostate / Q000473:pathology; D012965:Sodium Chloride / Q000737:chemistry; D013154:Spleen / Q000473:pathology; D013194:Staining and Labeling / Q000379:methods*; D013961:Thyroid Gland / Q000473:pathology
chemical_list: D005404:Fixatives; D000096:Acetone; D000431:Ethanol; D012965:Sodium Chloride; D005976:Glutaral