Figure 4
Plunge freezing followed by freeze-substitution with the hook-holding procedure. (a) Scheme illustrating the hook-holding procedure. To retain the morphology, we held the deep fascia and rectus femoris muscle with two hooks and grasping forceps, then excised it at 1 cm2 and plunged it into the cooled propane/isopentane mixture. Subsequently, plunge freezing followed by freeze-substitution was carried out. (b) Hematoxylin–eosin (HE) stain. In the first superficial layer, collagen fibers extend in various directions along with vessels replete with erythrocytes. The second intermediate layer (*) contains single straight and thick collagen fibers. The third deepest layer (#) is composed of straight and thin collagen fibers. Lightly stained loose connective tissue is observed between the second and third layers. Scale bar = 50 µm. (c) Elastika Van Gieson stain. Collagen bundles in the second intermediate layer are almost straight. Elastic fibers are observed in all layers of the deep fascia (arrowhead). Scale bar = 50 µm. (d) HE stain. The epimysium may exist independently of the third layer of the deep fascia and connected to the perimysium (arrowhead). Scale bar = 50 µm. (e) A quantitative comparison of the mean thicknesses of the deep fascia, first, second, and third layers, between the second and third layers, and the thickness ratio of the second layer to the deep fascia was conducted for 15 subjects per group. All data are presented as the mean ± standard deviation (SD). Statistical significance is determined using a Student’s t-test, with asterisks indicating statistically significant differences (*p < 0.05). NH, no hooks; HH, Hook-holding procedure; DF, deep fascia.
