Replica immunoelectron microscopic study of the upper surface layer in rat mandibular condylar cartilage by a quick-freezing method

Abstract

A quick-freezing and deep-etching method in combination with replica immunoelectron microscopy was applied for examining localization of hyaluronic acid and fibronectin on the upper surface layer of rat mandibular condylar cartilage. Rat temporomandibular joints were dissected with articular disks in order to leave the articular cartilage surface intact. The disks were slightly cut with razor blades for exposing the condylar articular cartilage surface. They were quickly frozen with the isopentane-propane cryogen (-193 degrees C) and prepared for freeze-fracturing and deep-etching replica membranes. They were additionally treated with 5% SDS and 0.5% collagenase to keep some antigens attached on the replica membranes. After such a treatment, a routine immunogold method was applied for clarifying the localization of hyaluronic acid and fibronectin in the upper surface layer. Small immunogold particles for hyaluronic acid were mainly localized around upper filamentous networks covered with amorphous materials, but large immunogold ones for fibronectin were localized on deep thicker fibrils. We have revealed the native architecture of the upper surface layer of mandibular condylar cartilage on the replica membranes and also three-dimensional localization of hyaluronic acid and fibronectin by the immunogold method.

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

“…93 Cells and fibrils appeared embedded in a matrix that we tentatively categorized as fibronectin-like (Fig. 11), based on its similarity to QFDE images of Zea-Aragon et al, 94 who identified similar structures via immunogold. Qualitatively, our QFDE image library showed that more of this fibronectin-like matrix was present in the 1% ITS þ 1% FBS culture (not shown).…”

Immunohistochemical Application of Cryotechniques to Native Morphology of Cells and Tissues

“…At an electron microscopic level, when the cryotechnique is combined with the freeze-fracture replica method as described in the previous section (Section II), we recently reported that the cryotechnique is applicable for the in situ immunolocalization of some soluble matrix molecules, such as hyaluronic acid and fibronectin, in the rat mandibular joint cartilage [20]. Therefore, we have now proposed that the quick-freeze replica method can maintain the original distribution of all biological molecules in cells and tissues by sealing them in a vitreous ice crystal environment.…”

Detection of injected fluorescence-conjugated IgG in living mouse organs using “in vivo cryotechnique” with freeze-substitution

“…This technique was performed by rapid freezing of these tissues with liquid isopentane-propane cryogen (À1938C) with or without cutting using a cryoknife (À1968C) cooled down in liquid nitrogen. In previous studies, the dynamic morphological changes in living cells, tissues, and organs using the cryotechnique were observed by electron and light microscopy (Ohno et al, 1996;Takayama et al, 1999Takayama et al, , 2000Terada et al, 1998;Xue et al, 2001;Zea-Aragon et al, 2004b).…”