Immunohistochemical Staining for Lymphatic and Blood Vessels in Normal Tissues: Comparison between Routinely Paraffin-Embedded Tissues and Frozen Sections

Abstract

<p><strong>Objective</strong>. In the current study, we compared the distribution of blood and lymphatic vessels from paraffin-embedded tissues with those of frozen tissues of normal human and rhesus monkey.</p><p><strong> Materials and Methods</strong>. We performed immunocytochemical staining for lymphatic and blood vessels using LYVE-1 for lymphatic vessels and von Willebrand factor (F-8) for blood ves- sels.</p><p><strong>Results</strong>. Normal tissues included spleen, lymph node, liver, pancreas, salivary gland, colon, diaphragm, heart, lung, thyroid, adrenal gland, kidney, ovary, endometrium, and prostate. Splenic sinusoids were stained for LYVE-1 and F-8 in the frozen sections, supporting that the sinusoid is a lymphoreticular system and blood vessel in structure and function. In frozen sections, the lymphatic sinusoids were consistently positive for LYVE-1, while hepatic sinusoids were positive for LYVE-1, but not for F-8. Thus, lymphatic and blood vessels were more readily detected in frozen tissue sections than in the paraffin-embedded sections. In the endometrium, lymphatic vessels were not diffusely immunostained in paraffin-embedded sections. However, frozen sections detected cyclic changes of lymphatic vessels, growing from basalis to functionalis in the menstrual cycle. Lymphatic vessels were immunostained in many organs using frozen sections. Small pulmonary blood vessels were not immunostained by F-8 in the periphery of the bronchial vessel tree most likely these smallest blood vessels were not immunostained due to less F-8 attached to their endothelia. Conclusion. The present findings illustrate the differences in the immunostaining of blood vessels in sections obtained from paraffin-embedded tissues and those from frozen tissue. These new findings may be relevant for the basic histology and histopathology of lymphatic and blood vessels.</p>

Comparative effects of hepatocyte growth factor and tacrolimus on acute liver allograft early tolerance

“…Liver graft, splenic, and kidney tissues were either cryoprotected in 30% sucrose overnight and frozen in optimal cutting temperature medium for IF staining ( 25 ) or embedded in paraffin ( 26 ) for IHC staining.…”

Immunohistochemical staining for nerve fibers in normal tissues with frozen sections: Structure and Function Relationship

“…Additional monkeys were inserted with Silastic IUD (6 cm) into uterine cavity filled with an IUD containing progesterone antagonist, ZK 23011 (Leiras OY, Finland), which was kept for 5 months 28 . After the normal organs and uterus had been removed, a wedge of tissue from the inner endometrial surface to the myometrium including the full thickness of endometrium and contiguous myometrium was taken as described before at an average tissue size of 1 x 1 x 0.4 cm [12][13][14] . Fresh wedge tissues were microwave-irradiated for 7 sec in a microwave oven, embedded in OCT, frozen in liquid propane in the liquid nitrogen bath and frozen sectioned at 5 -7 µm [12][13][14] .…”

“…After the normal organs and uterus had been removed, a wedge of tissue from the inner endometrial surface to the myometrium including the full thickness of endometrium and contiguous myometrium was taken as described before at an average tissue size of 1 x 1 x 0.4 cm [12][13][14] . Fresh wedge tissues were microwave-irradiated for 7 sec in a microwave oven, embedded in OCT, frozen in liquid propane in the liquid nitrogen bath and frozen sectioned at 5 -7 µm [12][13][14] . Frozen sections were mounted on Super Frost Plus slide (Fisher Scientific, Pittsburgh, PA), microwave-irradiated again on ice for 3 sec, fixed in 2% paraformaldehyde in phosphate buffer at pH 7.4 for 10 to15 min at room temperature, and immersed twice for 2 min each in 85% ethanol [12][13][14] .…”

“…Fresh wedge tissues were microwave-irradiated for 7 sec in a microwave oven, embedded in OCT, frozen in liquid propane in the liquid nitrogen bath and frozen sectioned at 5 -7 µm [12][13][14] . Frozen sections were mounted on Super Frost Plus slide (Fisher Scientific, Pittsburgh, PA), microwave-irradiated again on ice for 3 sec, fixed in 2% paraformaldehyde in phosphate buffer at pH 7.4 for 10 to15 min at room temperature, and immersed twice for 2 min each in 85% ethanol [12][13][14] . Sections were incubated with blocking serum for 20 min and then with monoclonal anti-human NF (clone 2F11) and monoclonal CD56 (Dako System, Carpenteria, CA) at 1:100 dilutions overnight at 4°C, respectively.…”

“…Skin biopsy for the assessment of intradermal sensory nerve fiber using the pan-axonal marker, PGP 9.5 is an established clinical and research technique, and this technique may also be used to assess other population of nerve fibers in the other tissues [8][9][10][11] . We had previously utilized immunohistochemical staining with frozen sections for immunostaining lymphatic and blood vessels with normal monkey tissues 12,13 and extended this staining technique to immunostain peripheral nerve fibers in the normal tissues 14 . The current study aimed to detect the presence of nerve fibers in normal tissues including heart, lung, small intestine, large intestine, diaphragm, spleen, pancreas, adrenal gland, kidney, urinary bladder, vagina and normally cycling endometrium.…”