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Fixation and Sectioning

Fixation and Sectioning

Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation is the wet mount, in which the specimen is placed in a drop of liquid on the slide. A liquid specimen can be directly deposited on the slide using a dropper. Solid specimens, such as skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid is simply water, but stains are often added...

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Related Experiment Video

Updated: May 23, 2026

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

Published on: March 25, 2020

Optimal antigen retrieval for ethanol-fixed cytologic smears.

Tamami Denda¹, Shingo Kamoshida, Jumpei Kawamura

¹Division of Diagnostic Pathology and Cytology, Higashiyamato Hospital, Nangai, Higashiyamato, Tokyo, Japan. t.denda@yamatokai.or.jp

Cancer Cytopathology

|March 22, 2012

Summary

Antigen retrieval is often unnecessary for ethanol-fixed smears in immunostaining. Mild heat-induced retrieval with citrate buffer (pH 6.0) is optimal for most antibodies, but individual optimization is key.

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Area of Science:

Histopathology
Immunohistochemistry
Cytopathology

Background:

Antigen retrieval is vital for immunostaining formalin-fixed, paraffin-embedded (FFPE) tissues.
Its necessity for ethanol-fixed smears remains largely uninvestigated.
This study assesses heat-induced and protease-induced antigen retrieval on ethanol-fixed smears.

Purpose of the Study:

To evaluate the efficacy of antigen retrieval methods for immunostaining ethanol-fixed smears.
To compare staining results between ethanol-fixed smears and FFPE tissue sections.
To determine optimal antigen retrieval conditions for various antibodies on ethanol-fixed smears.

Main Methods:

Immunostaining of 43 antibodies on 36 Papanicolaou-stained ethanol-fixed smears.
Application of citrate buffer (pH 6.0, 7.0) and EDTA (pH 8.0) for heat-induced retrieval.
Use of pronase for protease-induced retrieval, with comparison to FFPE sections.

Main Results:

Heat-induced antigen retrieval was crucial for nuclear antigens and some cytoplasmic/membrane antigens.
Lower pH citrate buffer (pH 6.0) proved superior for ethanol-fixed smears.
Many antibodies stained well without retrieval on ethanol-fixed smears, unlike FFPE sections.
Protease-induced retrieval often harmed ethanol-fixed smears; formalin vapor fixation aided detection of some markers.

Conclusions:

Most antibodies can be immunostained on ethanol-fixed smears without retrieval or with mild heat-induced retrieval (citrate buffer, pH 6.0).
Optimal antigen retrieval conditions require individual determination for each antibody.
Ethanol fixation offers an alternative for certain immunostaining applications.