IngentaConnect Cytochemical investigation of gastric gland component cells with ...

Authors: Tsuyama, Shinichiro¹; Matsushita, Sachi¹; Takatsuka, Tomio¹; Nonaka, Satoru²; Hasui, Kazuhisa¹; Murata, Fusayoshi¹

Source: Anatomical Science International, Volume 77, Number 1, March 2002 , pp. 73-82(10)

Publisher: Blackwell Publishing

Abstract:

Gastric gland component cells were electron-microscopically and immunoelectronmicroscopically examined with high-pressure freezing followed by freeze substitution and a low-temperature embedding resin method and compared to that of the conventional chemical-fixation method. The rat gastric gland was high-pressure frozen, freeze-substituted with acetone-containing osmium or acrolein, and embedded in Epon 812 or Lowicryl K4M, respectively. Using the high-pressure freezing method, the vitreous freezing range reached the depth of 150 µm from the surface. The ultrathin sections from both procedures embedding in Epon 812 and Lowicryl K4M were doubly stained with uranyl acetate and lead acetate, and histochemically or immunohistochemically stained, respectively. In comparison to the conventional chemical fixation method, excellent results were obtained with respect to ultrastructural preservation. The stainings performed in this experiment included periodic acid-thiocarbohydrazide-silver proteinate staining, cationic colloidal cold at pH 2.5 staining, Helix pomatia lectin-staining, anti-alpha or -beta subunit antibodies of H⁺K⁺-ATPase immunostaining and pepsinogen immunostaining. The staining intensity of those was stronger than that of the conventional immersion-chemical fixation method. In addition to these results, the labels also showed good specific localization. In this paper, we provide a description of the high-pressure freezing followed by freeze substitution and low-temperature embedding resin method compared to the conventional chemical-fixation method. Our results suggest that this method is a suitable tool for ultrastructural and histochemical/immunohistochemical studies at high resolution.

Keywords: component cell; freeze substitution; gastric gland; glycoconjugate histochemistry; high-pressure freezing

Document Type: Research article

DOI: 10.1046/j.0022-7722.2002.00011.x

Affiliations: 1: Department of Anatomy and 2: Center of Electron Microscopy, Kagoshima University, Faculty of Medicine, Kagoshima, Japan

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Cytochemical investigation of gastric gland component cells with high-pressure freezing followed by freeze-substitution and hydrophilic resin embedding