A General Method for the Specific Staining of Intracellular Antigens with Ferritin-Antibody Conjugates

McLean, Jaime; Singer, S. J.

doi:10.1073/pnas.65.1.122

Cited by 80 publications

(27 citation statements)

References 12 publications

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“…To be successful, these techniques require preservation of antigenicity of intracellular proteins, accessibility of these proteins to ferritin-conjugated antibody, and retention of cell ultrastructure during experimental manipulation. In one method (15,20), the tissue was embedded in albumin and sections (approximately 600 A) exposed to antibody, whereas in the other (23), prefixed, broken cells were exposed to antibody before embedding by routine methods. In both, affinity-purified, ferritin-conjugated antibody provided a specific marker for antigen location.…”

mentioning

confidence: 99%

Location of procollagen in chick corneal and tendon fibroblasts with ferritin-conjugated antibodies.

Nist¹,

Mark²,

Hay³

et al. 1975

The Journal of Cell Biology

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Three distinct antiprocollagen preparations were characterized and used in immunocytochemicai staining of chick embryo corneal and tendon cells. The several ferritin-conjugated antibody preparations permitted similar location of procollagen in the cisternae of the rough endoplasmic reticulum and in Golgi elements in both cell types. The ability to demonstrate and interpret specific ferritin staining was dependent on the extent of membrane breakage in each of these organelles, coupled with adequate retention of cell morphology. Corneal fibroblasts appeared to suffer more extensive intracellular membrane damage under controlled conditions of homogenization than tendon fibroblasts, facilitating the identification of procollagen in Golgi vacuoles of these cells. None of the labeled material appeared to be cytoplasmic in origin since ferritin was observed in the cytoplasm only in the vicinity of Golgi elements that were extensively broken. This study extends previous immunological evidence for the presence of procollagen in the Golgi complex and calls attention to the problems to be encountered in locating the antigen in small Golgi vesicles and lamellae.

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mentioning

confidence: 99%

Location of procollagen in chick corneal and tendon fibroblasts with ferritin-conjugated antibodies.

Nist¹,

Mark²,

Hay³

et al. 1975

The Journal of Cell Biology

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“…24,25 All of the protein-based solutions we examined here facilitated to some extent cryosectioning of the PEG constructs, but only 30% BSA and 100% FBS were able to reproducibly retain the construct integrity. The formation of voids within cross-sections suggests that incomplete construct infiltration was a common problem, which might depend on the category and the concentration of solutes in the infiltration solutions, as well as the duration of incubation.…”

Section: Discussionmentioning

confidence: 99%

An Improved Cryosection Method for Polyethylene Glycol Hydrogels Used in Tissue Engineering

Ruan

Tulloch

Muskheli

et al. 2013

Tissue Engineering Part C: Methods

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The high water content of hydrogels allows these materials to closely mimic the native biological extracellular conditions, but it also makes difficult the histological preparation of hydrogel-based bioengineered tissue. Paraffin-embedding techniques require dehydration of hydrogels, resulting in substantial collapse and deformation, whereas cryosectioning is hampered by the formation of ice crystals within the hydrogel material. Here, we sought to develop a method to obtain good-quality cryosections for the microscopic evaluation of hydrogelbased tissue-engineered constructs, using polyethylene glycol (PEG) as a test hydrogel. Conventional sucrose solutions, which dehydrate cells while leaving extracellular water in place, produce a hydrogel block that is brittle and difficult to section. We therefore replaced sucrose with multiple protein-based and nonprotein-based solutions as cryoprotectants. Our analysis demonstrated that overnight incubation in bovine serum albumin (BSA), fetal bovine serum (FBS), polyvinyl alcohol (PVA), optimum cutting temperature (OCT Ò ) compound, and Fisher HistoPrep frozen tissue-embedding media work well to improve the cryosectioning of hydrogels. The protein-based solutions give background staining with routine hematoxylin and eosin, but the use of nonprotein-based solutions PVA and OCT reduces this background by 50%. These methods preserve the tissue architecture and cellular details with both in vitro PEG constructs and in constructs that have been implanted in vivo. This simple hydrogel cryosectioning technique improves the methodology for creation of good-quality histological sections from hydrogels in multiple applications.

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“…The ultrathin frozen sections (2,7,12,13,18) and the BSAembedding method (6,10) are fit for occasional purposes, but these methods are not always generalized, because the ultrastructure of the cell and tissue is not well preserved by the former method and the preparation of ultrathin sections is quite difficult in both methods.…”

Section: Discussionmentioning

confidence: 99%

“…Ultrathin frozen sections are used in some immunohistochemical studies (2,7,12,13,18), but destruction of the fine structure and the release of antigens occur during the preparation of the ultrathin frozen sections. The embedding method by using crosslinked bovine serum albumin (BSA-embedding method) is sometimes useful (6,10), but it cannot be a generalized method, because it is difficult to prepare the ultrathin sections, and the antigenicities of the antigens in the embedded tissues are not always well preserved as the result of using a high concentration of cross-linking reagents (glutaraldehyde or paraformaldehyde) during polymerization. So, it is necessary for the immunohistochemical study to devise a new embedding method which can fill the following needs; 1) A minimal loss of antigenicities during the embedding process, 2) A simple preparation of ultrathin sections requiring no special devices, and 3) Removal of embedding materials from sections can be done under mild conditions.…”

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New Embedding Method for Immunohistochemical Studies Using Acrylamide Gel

Yamamoto

Yamashita

Yasuda

1980

Acta Histochem. Cytochem

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This study was attempted in order to devise a new embedding method for immunohistochemical studies to meet the following demands: 1) Minimum loss of antigenicities during the embedding process.2) Simple preparation of ultrathin sections requiring no special devices.3) Removal of embedding materials from sections under mild conditions. Acrylamide gel, cross-linked with acrylylcystamine or N,N'-diallyltartardiamide, proved to be a useful embedding media for immunocytochemistry in both light-and electron microscopy.For immunohistochemical study on the electron microscopic level it is desirable that the labeled antibody reacts directly with the antigens on the cut-face of the ultrathin sections, because the labeled antibody cannot penetrate evenly into the fixed tissue block (19). Except in special cases (5), the conventional embedding materials for electron microscopic study, such as epoxy resin and methacrylate resin, cannot be applied to immunohistochemical study for the following two reasons: 1) Most of the antigens lose their antigenicities during the embedding process of tissues, 2) The removal of embedding resin from the sections is not easy. Ultrathin frozen sections are used in some immunohistochemical studies (2,7,12,13,18), but destruction of the fine structure and the release of antigens occur during the preparation of the ultrathin frozen sections. The embedding method by using crosslinked bovine serum albumin (BSA-embedding method) is sometimes useful (6, 10), but it cannot be a generalized method, because it is difficult to prepare the ultrathin sections, and the antigenicities of the antigens in the embedded tissues are not always well preserved as the result of using a high concentration of cross-linking reagents (glutaraldehyde or paraformaldehyde) during polymerization. So, it is necessary for the immunohistochemical study to devise a new embedding method which can fill the following needs; 1) A minimal loss of antigenicities during the embedding process, 2) A simple preparation of ultrathin sections requiring no special devices, and 3) Removal of embedding materials from sections can be done under mild conditions. This study was attempted in order to develop a new embedding method suitable *

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A General Method for the Specific Staining of Intracellular Antigens with Ferritin-Antibody Conjugates

Cited by 80 publications

References 12 publications

Location of procollagen in chick corneal and tendon fibroblasts with ferritin-conjugated antibodies.

Location of procollagen in chick corneal and tendon fibroblasts with ferritin-conjugated antibodies.

An Improved Cryosection Method for Polyethylene Glycol Hydrogels Used in Tissue Engineering

New Embedding Method for Immunohistochemical Studies Using Acrylamide Gel

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