An Application of Cryofixation to Immunocytochemistry

Atsushi Ichikawa, Misao Ichikawa, Kenjiro Yasuda, Shuji Yamashita, Sadakazu Aiso, Masahide Shiozawa

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

An applicable method of cryofixation to immunocytochemistry was examined. Fresh tissue blocks of rat pancreas and parotid gland were quickly frozen by the metal contact method using liquid helium and freeze-substituted with one of the following media kept at —80°C for 36 hr; pure acetone, 0.1% glutaraldehyde (anhydrous) in acetone, approximately 0.2% paraformaldehyde in acetone, and 10% acrolein in acetone. After freeze-substitution fixation, tissue blocks were embedded in Araldite mixture. Thin sections mounted on nickel grids were processed for immunocytochemical localization of amylase according to the multiplestep protocol of the protein A-gold immunostaining method by Bendayan and Duhr (4). They were then postfixed with 2.5% glutaraldehyde in PBS and stained with uranyl acetate and lead citrate for electron microscopy. Good results were obtained from the materials substituted with glutaraldehyde or paraformaldehyde in acetone. The ultrastructural features of the cells were preserved well, similar to those in the materials substituted with OsO4 in acetone except for negative images of the membranous structures. Secretory granules, condensing vacuoles, and Golgi cisterns were labeled well with immunogold. Labeling density was much higher in the present materials than in those processed by conventional chemical fixation, and the intensity of labeling increased in proportion to increasing electron density of the materials contained within individual subcellular compartments. These results indicate that an application of the cryofixation method is useful for improving resolution and specificity in immunocytochemical postembedding staining.

Original languageEnglish
Pages (from-to)601-609
Number of pages9
JournalActa Histochemica et Cytochemica
Volume20
Issue number6
DOIs
Publication statusPublished - 1987 Jan 1
Externally publishedYes

Fingerprint

Acetone
Glutaral
Labeling
Tissue
Acrolein
Helium
Staphylococcal Protein A
Amylases
Nickel
Citric Acid
Gold
Contacts (fluid mechanics)
Electron microscopy
Carrier concentration
Immunohistochemistry
Rats
Substitution reactions
Metals
Liquids

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biochemistry
  • Physiology
  • Histology
  • Cell Biology

Cite this

Ichikawa, A., Ichikawa, M., Yasuda, K., Yamashita, S., Aiso, S., & Shiozawa, M. (1987). An Application of Cryofixation to Immunocytochemistry. Acta Histochemica et Cytochemica, 20(6), 601-609. https://doi.org/10.1267/ahc.20.601

An Application of Cryofixation to Immunocytochemistry. / Ichikawa, Atsushi; Ichikawa, Misao; Yasuda, Kenjiro; Yamashita, Shuji; Aiso, Sadakazu; Shiozawa, Masahide.

In: Acta Histochemica et Cytochemica, Vol. 20, No. 6, 01.01.1987, p. 601-609.

Research output: Contribution to journalArticle

Ichikawa, A, Ichikawa, M, Yasuda, K, Yamashita, S, Aiso, S & Shiozawa, M 1987, 'An Application of Cryofixation to Immunocytochemistry', Acta Histochemica et Cytochemica, vol. 20, no. 6, pp. 601-609. https://doi.org/10.1267/ahc.20.601
Ichikawa A, Ichikawa M, Yasuda K, Yamashita S, Aiso S, Shiozawa M. An Application of Cryofixation to Immunocytochemistry. Acta Histochemica et Cytochemica. 1987 Jan 1;20(6):601-609. https://doi.org/10.1267/ahc.20.601
Ichikawa, Atsushi ; Ichikawa, Misao ; Yasuda, Kenjiro ; Yamashita, Shuji ; Aiso, Sadakazu ; Shiozawa, Masahide. / An Application of Cryofixation to Immunocytochemistry. In: Acta Histochemica et Cytochemica. 1987 ; Vol. 20, No. 6. pp. 601-609.
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