Connective Tissue Ultrastructure: A Direct Comparison between Conventional Specimen Preparation and High‐Pressure Freezing/Freeze‐Substitution

Abstract

It is generally agreed within the microscopy community that the quality of ultrastructure within the connective tissue matrix resulting from high-pressure freezing followed by freeze-substitution (HPF/FS) far exceeds that gained following the "conventional" preparation method, which includes aqueous fixation, dehydration, and embedding. Exposure to cryogen at high pressure is the only cryopreservation method capable of vitrifying tissue structure to a depth exceeding 200 μm. Cells within connective tissues prepared by HPF/FS are universally larger, filling the commonly seen void at the juncture between cell and matrix. Without significant shrinkage of cells and the coincident extraction of the cytosolic components, well-resolved organelles are less clustered within an expanded cytosol. Much of the artifact from "conventional" methods occurs as large space filling and also smaller fibrilassociated proteoglycans are extracted during fixation. However, the visualization of some matrix features by electron microscopy is actually dependent on the collapse or extraction of these "masking" components. Herein, we argue that an impression of ultrastructure within commonly studied matrices, in particular skin, is best gained following the evaluation of both conventional preparations and tissue prepared by HPF/FS. Anat Rec, 303:1514Rec, 303: -1526Rec, 303: , 2020 Microscopists have traditionally relied on aqueous fixation, dehydration, and embedding in resin (herein referred to as the "conventional" preparation) for the evaluation of extracellular matrix (ECM) ultrastructure. It was in the mid-1960s when Farquhar and Palade (1965) combined the use of glutaraldehyde followed by osmium tetroxide

Vertebrate extracellular matrix protein hemicentin-1 interacts physically and genetically with basement membrane protein nidogen-2

“…Finally, grids were rinsed in Tris for 15 minutes, then in distilled water for 15 minutes. Sections were evaluated unstained for background and specific labeling, then contrasted for 4 minutes in 4% Uranyl acetate followed by quarter-strength Reynold’s lead citrate for 15 seconds (91).…”

“…Finally, grids were rinsed in Tris for 15 minutes, then in distilled water for 15 minutes. Sections were evaluated unstained for background and specific labeling, then contrasted for 4 minutes in 4% Uranyl acetate followed by quarter-strength Reynold's lead citrate for 15 seconds(72).StatisticsQuantitative experiments were repeated at least three times, reaching similar results. Mean values and standard deviations of all individual specimens from one representative or all independent experiments are presented, as specified in the respective figure legends.…”

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“…Until recently, in traditionally processed tissues for transmission electron microscopy (TEM) (including fixation in glutaraldehyde or formaldehyde) [1] it was considered that the basement membrane (BM) consists of two overlapping well-defined layers: the lamina lucida, a homogeneous structure, clear in electrons flow, and the lamina densa [2], with anchoring filaments, that are extending from the hemidesmosomes (HDs) through the lamina lucida [1]. The preparation techniques by high-pressure freezing (HPF) method and subsequent freeze substitution (FS) can preserve a greater tissue integrity and therefore, it has been found that lamina lucida is actually absent and represents only a fixation artifact.…”

The Good, the Bad and the Ugly: Task-Specific Fixation for Connective Tissues

“…HPF/FS is triumphant in revealing the ultrastructure of smaller organisms; however the successes in cooling exceedingly hydrated tissue ice-crystal free are so rare that biopsies must not be sacrificed to this procedure. Certainly, the few images of well cryo-preserved connective tissue need consideration when deliberating spatial relationships in this difficult tissue (1).…”