The Preservation of Fine Structure by Use of Dimethylsuberimidate as a Fixative

Ono, Tetsuro; Yamamoto, Noboru; Yasuda, Kenjiro

doi:10.2535/ofaj1936.53.4_199

Cited by 4 publications

(2 citation statements)

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“…[5] Moreover, DTBP stood out since it was known in electron microscopy studies for its remarkable ability to preserve the fine filamentous or microtubular structures of the cell membrane. [6] Tunneling nanotubes (TNTs) are cellular extension notoriously fragile to fixation and therefore can be used to measure the cells’ structural integrity. While glutaraldehyde (GLU)-based fixation (GLU/PFA) is ideal to maintain TNTs [7] and therefore structural integrity (Figure 1B), it can hinder downstream MS analysis.…”

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A Novel Cell Fixation Method that Greatly Enhances Protein Identification in Microproteomic Studies Using Laser Capture Microdissection and Mass Spectrometry

2018

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Microproteomic studies have improved our knowledge of cell biology. Yet, with mass spectrometry (MS) analysis, accuracy can be lost for protein identification and quantification when using heterogeneous samples. Laser capture microdissection (LCM) allows for the enrichment of specific subsets of cells to study their proteome; however, sample fixation is necessary. Unfortunately, fixation hampers MS results due to protein cross-linking. The aim of this study was to identify both a fixation protocol and an extraction method that returns the best yield of proteins for downstream MS analysis, while preserving cellular structures. We compared glutaraldehyde (GLU), a common fixative to preserve cells, to dithiobispropionimidate (DTBP), a cleavable cross-linker. Our DTBP fixation/extraction protocol greatly increased the protein recovery. In fact, while 1000 GLU fixed cells returned only 159 unique protein hits, from 1464 unique peptides of 1994 unique collected spectra, 1000 DTBP fixed cells resulted in 567 unique collected protein hits, from 7542 unique peptides, of 10,401 unique collected spectra. That is, a 3.57-fold increase in protein hits, 5.15-fold increase in unique peptides, and a 5.22-fold increase in unique collected spectra. Overall, the novel protocol introduced here allows for a very efficient protein recovery and good sample quality for MS after sample collection using LCM.

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A Novel Cell Fixation Method that Greatly Enhances Protein Identification in Microproteomic Studies Using Laser Capture Microdissection and Mass Spectrometry

2018

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“…By using two other reagents as a fixative (diimidoesters and water soluble carbodiimide) the fine structure was as well preserved as when glutaraldehyde was employed. The preservation o f enzymatic activities after fixation with aldehyde, diimidoester (DAIS), water soluble carbodiimide (WSC) and cyanuric chloride (Yamamoto, 1975 ;Ono et al, 1976 ;Yamamoto et al , 1976) . These results suggest that these three cross-linking reagents are useful as the primary fixative in electron microscopic study.…”

Section: ) Fixatives Suitable For Histochemical As Well As Immunohismentioning

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Application of Metal Labeled Antibody Method and a Consideration of the Fixatives Suitable for Immunohistochemical Study

Yamamoto

1977

Acta Histochem. Cytochem

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1) The preparation of metal labeling antibody;Since Singer (1959) introduced the immunoferritin method, many metals have been used for labeling antibody. Metal labeled antibody has an advantage over other labeled antibodies both in its smaller molecular weight and in producing a sharp image under the electron microscope at high magnification. However, the number of metal atoms capable of conjugating with the antibody without losing antibody reactivity is quite limited, and this restriction leads to the production of insufficient electron opacity. In order to both minimize the loss of antibody titer caused by the direct labeling of heavy metal to antibody and to label as many metal atoms as possible to the antibody molecule to obtain high contrast, an indirect label of 1-(chloromercuri) ferrocene (CFM) to antibody was introduced, with thiolated egg albumin (EA) employed as an intermediate material. The method was reported in detail elsewhere (Yasuda and Yamamoto, 1975). Fig. 1 shows the location of pancreatic amylase in the zymogen granule of the acinal cell of mouse pancreas stained by CMF-labeled antibody. Many tiny grains of almost uniform size representing the location of amylase are recognized in the zymogen granules, while the grains were scarcely observed in the area outside of the zymogen granules. The concentration of the grains within the zymogen granule demonstrated by this method was heavier than that demonstrated by use of the ferritin-labelled antibody. This difference may be due to the difference in the molecular weight of the labelling materials ; The molecular weight of CMF-EA (MW. of CMF; 421; EA MW. of 45,000) is much smaller than that of ferritin (MW. 650,000 on the average). Judging from this result, this new labelling method would seem to be useful for immunohistochemical study, especially for the observation of the localization of antigens at high magnification in electron microscopy. 2) Preparation of specimens for immunohistochemistry;It is a question whether or not protein molecules such as y-globulin or its Fab fragments labeled by any kind of substance can penetrate to the cell membrane of fixed cells. As a test examination conducted in order to determine the ratio of protein penetration into the tissue block, horseradish peroxidase (MW. 39,000) was chosen as the test material in this study. Mouse liver was cut into small blocks of approximately 1 mm2 X 5 mm in size and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 hr, and then washed overnight with the same buffer containing 5 % sucrose. Non-frozen sections, about 40-50 , i in thickness, were prepared with a Smith-Farquhar tissue sectioner (Iran Sorvall). The

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Immunologische und enzymatische Aktivität nach Fixierung mit Diimidoester Verbindungen

et al. 1980

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The Preservation of Fine Structure by Use of Dimethylsuberimidate as a Fixative

Cited by 4 publications

References 29 publications

A Novel Cell Fixation Method that Greatly Enhances Protein Identification in Microproteomic Studies Using Laser Capture Microdissection and Mass Spectrometry

A Novel Cell Fixation Method that Greatly Enhances Protein Identification in Microproteomic Studies Using Laser Capture Microdissection and Mass Spectrometry

Application of Metal Labeled Antibody Method and a Consideration of the Fixatives Suitable for Immunohistochemical Study

Immunologische und enzymatische Aktivität nach Fixierung mit Diimidoester Verbindungen

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