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Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures
Published on: April 3, 2013
Immunolabelling of prolactin at ultrastructural level using the protein A-gold technique on Epon-embedded tissue
1Department of Radiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Insights
This study introduces a new post-embedding technique for electron microscopy to precisely locate prolactin in human pituitary cells. This method aids in understanding hormonal secretion disorders at a detailed cellular level.
Area of Science:
- Cell Biology
- Endocrinology
- Immunocytochemistry
Background:
- Colloidal-gold techniques are vital for localizing intracellular antigens in electron microscopy.
- Accurate localization of hormones like prolactin is crucial for understanding pituitary function and disease.
Purpose of the Study:
- To develop and validate a post-embedding immunogold technique for prolactin localization in resin-embedded human pituitary tissue.
- To enable ultrastructural studies of prolactin secretion and its role in pathophysiology.
Main Methods:
- Human pituitary tissue was processed for electron microscopy without post-osmication and embedded in Epon.
- An indirect immunoperoxidase method was used for light microscopic identification of lactotroph cells.
- Ultra-thin sections were labeled with anti-prolactin antibodies followed by protein-A gold conjugate.
Main Results:
- Specific prolactin labeling was observed on secretory granules.
- Labeling density ranged from 15-30 particles per granule, quantified using the Quantimet 570 image analysis system.
- The technique demonstrated high specificity for intracellular prolactin localization.
Conclusions:
- The developed post-embedding technique allows for precise ultrastructural localization of prolactin in human pituitary tissue.
- This method is a valuable tool for investigating the pathophysiology of hormonal secretion and for diagnostic research.
Abstract:
The use of the colloidal-gold technique in electron microscopy immunocytochemistry has provided important information on the in situ localisation of intracellular antigens. We have developed a post-embedding technique for prolactin localisation on resin-embedded human pituitary tissue sections by the use of the protein-A gold conjugate. Human pituitary tissue obtained at autopsy was processed for electron microscopical study without post-osmication and then embedded in Epon. The indirect immunoperoxidase method was used for light microscopical targetting of lactotroph cells for subsequent electron microscopical antigen localisation. Ultra-thin sections were labelled with human anti-human prolactin followed by protein-A gold conjugate. Specific labelling was observed over secretory granules with a density of 15-30 particles per granule, as determined by the Quantimet 570 image analysis system. This technique provides a means of studying the pathophysiology of hormonal secretion at ultrastructural level and can be a useful tool in diagnostic and research investigations.
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