Biomedical subjects

B Kniola

Publications and source records attributed to B Kniola.

5 recordsLinked to original sources

PubMed1992

Epon resin infiltration and immunogold labelling of pituitary secretory granules after cryofixation versus chemical fixation.

The degree of infiltration of epoxy resin into pituitary secretory granules was evaluated using X-ray microanalysis of the concentrations of chlorine in the epoxy resins. The effectiveness of infiltration was tested after three different tissue preparation techniques: cryofixation+freeze-drying (CF-FD), glutaraldehyde fixation (GF)+chemical dehydration, and no fixation--no dehydration. Signs of marked incomplete infiltration were found in embedded unfixed tissue while the other two techniques showed 80% infiltration. Uneven penetration was seen after CF-FD and GF. The plastic surface demonstrated a mountain-like appearance over the secretory granules after immunocytochemistry of the glutaraldehyde fixed tissue, whereas the CF-FD tissue showed a less furrowed surface. This probably is due to contact with water, which swells those parts of the granules that are unprotected by the plastic embedding medium. Our findings may explain why it is possible to perform immunocytochemistry on Epon embedded tissue.

Animals↗

PubMed1992

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.

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.

Animals↗

PubMed1991

Pituitary secretory granule topography: influence of different electron microscopic preparation procedures on the surface of Epon sections.

Human, rat and mouse pituitary tissues have been examined electron microscopically in transmission (TEM), scanning-transmission (STEM) and scanning (SEM) modes for the surface appearance of the secretory granules in tissue sections. Cryofixed and cryosectioned tissue showed only slightly protruding granule profiles which had a smooth surface. Cryofixed, freeze-dried and Epon embedded pituitaries, on the other hand, demonstrated swollen and furrowed surfaces over the granules after contact with water. This topography could also be seen after glutaraldehyde fixation but less after post-fixation in OsO4. The surface alterations in the sections of pituitary secretory granules are thought to be due to differences in the homogeneity of the resin infiltration, leaving resin-free openings where water can enter. It also seems probable that the Epon resin is more influenced by water than has been previously assumed, based on the findings of efficient elimination of osmium from the granules after incubation of tissue sections in water for only 10 min.

Animals↗

PubMed1990

Quantitative ultrastructural immunocytochemistry using a computerized image analysis system.

Secretory granules in human pituitary adenoma cells have been examined indirectly for hormone epitopes by immunogold labelling of resin-embedded ultrathin sections. The specific binding of different immunoglobulin-gold complexes to the antigrowth hormone antibodies over the secretory granules was measured using a computerized image analysis system. This facilitated the assessment of the preferential binding to the target granules of gold particles with three different average particle diameters (Au7, Au11, Au17). The time of pretreatment of sections with H2O2 or a buffer was found to influence the staining considerably. The scanning electron microscopic findings of protruded secretory granules with a mountain-like surface might be relevant to the uneven distribution of immunolabels seen over the secretory granules in the adenohypophysis.

Adenoma↗

PubMed1988

Reduced redox potential during growth of some gram-negative bacteria. Effect on the biochemical and physicochemical surface properties and phagocytosis by polymorphonuclear leukocytes.

When cultivated at reduced redox potential the physico-chemical surface properties were altered in strains of E. coli, Salmonella and Yersinia bacteria. In particular, strains which showed hydrophilic surface properties under normal aerobic cultivation became more hydrophobic when exposed to anaerobic conditions (e.g. E. coli K12, E. coli K12D21, E. coli K12D22, S. minnesota S99, S. typhimurium 395MS, S. braenderup 2828 and Yersinia enterocolitica). Moreover, there were qualitative as well as quantitative differences in the protein profiles of whole bacterial lysates and membrane preparations analysed in SDS-PAGE. There were no qualitative differences in the lipopolysaccharide (LPS) bands. However, when E. coli K12D22 were cultivated aerobically, remarkably more high molecular temperature-sensitive (70 degrees C for 45 min) carbohydrate material was produced (weight about 360 KD and 660 KD). Interaction between polymorphonuclear leukocytes (PMNL) and the E. coli K12D22 strain, measured as chemiluminescence, showed that the anaerobically cultivated bacteria induced a chemiluminescence that was mainly of intracellular origin, while the aerobically cultivated induced an extracellular response. Phagocytosis and killing-studies showed that only anaerobically-grown E. coli were effectively inactivated by the PMNL.

Aerobiosis↗