Short Fixation-Shock Freezing and Freeze-Drying versus Chemical Fixation and Dehydration: Computer Assisted Image Analysis of Morphological Variables and Immunogold Labeling Density on Pituitary Secretory Granules
作者:
EneströmSverker,
KniolaBarbara,
期刊:
Biotechnic&Histochemistry
(Taylor Available online 1992)
卷期:
Volume 67,
issue 5
页码: 268-287
ISSN:1052-0295
年代: 1992
DOI:10.3109/10520299209110036
出版商: Taylor&Francis
关键词: rat somatotrophs;nuclei;granules;specific and nonspecific immunolabeling;im-munostaining;computerized image analysis
数据来源: Taylor
摘要:
We have performed a computer assisted image analysis evaluation of the effects of two preparation protocols on morphological variables and immunolabeling density of secretory granules using rat adenohypophysis as a model. Glutaraldehyde (GA) fixation for 2 hr and chemical dehydration was compared with short GA fixation (15 min) followed by cryofixation and freeze-drying (CF-FD). The 2 hr GA fixed specimens showed spherical nuclei and secretory granules regardless of their size, contrasting with the more irregularly shaped nuclei and secretory granules after short GA-CF-FD. In the latter group of specimens a correlation could be found between smaller nuclear areas and more irregular shapes. The differences in morphological variables between the two preparation protocols might be due to a better protein stabilization and a reduced collapse of macromolecules after the 2 hr GA fixation, strengthened by the chemical dehydration. The specific immunolabeling with antiserum to growth hormone was much greater but more varied after short time GA-CF-FD than after long GA fixation. Epon surface topography over the granule area also differed: smooth after short time GA-CF-FD and furrowed after long GA fixation. Our results, demonstrating important differences in morphological parameters and immunolabeling density between two common preparation protocols, seem critical for more reliable interpretation in quantitative immunoelectron microscopy. We also emphasize the need for computer assisted image analysis and measurements in immunoelectron microscopy to ensure objective evaluations.
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