Surgically resected aortic tissue was fixed in 4% paraformaldehyde for 24 h then embedded in paraffin. Trimmed wax blocks were placed on a paraffin microtome (Thermo Fisher Scientific, Waltham, Uniteds States, HM325) by the pathology laboratory of Biossci (Hubei, China) Biotechnologies Company Ltd., and 4 μm-thick pieces were continuously cut. Sections were stained with elastin-van Gieson and immunofluorescence. Anti-elastin antibody (Abcam, ab213720, 1:1000) was used as primary antibody. Images were obtained using the HAMAMATSU imaging system (C13220-0, Japan). Measurements were performed three times.
Paraffin microtome
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Paraffin Microtome is a laboratory instrument used for cutting thin, uniform sections of paraffin-embedded tissue samples. It allows for the precise and consistent slicing of specimens for microscopic examination and analysis.
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5 protocols using paraffin microtome
1
Histological analysis of aortic tissue
2
Histological Processing of Colon Tissue
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The colon tissue was fixed with 10% neutral phosphate buffered formalin for 7 days. After washing with tap water, the tissues were sequentially placed in gradient ethanol by standard methods [12 (link)], and processed in xylene-ethanol mixture (45 min)-xylene (20–60 min)-melted paraffin at 70 °C (2 h). The embedded colon tissue were cut into 6 μm sections on Paraffin Microtome (Thermo, UK).
Slices were deparaffinized and rehydrated with standard methods, then processed with Hematoxylin staining for 5–10 min, differentiated with 0.5% hydrochloric acid ethanol for 30 s, and stained with 0.5% Eosin for 10 s, following by rinsing with tap water for 25 min. Finally, the slices were dehydrated with standard protocol and sealed.
For immunohistochemistry assay, slices were deparaffinized and rehydrated by standard methods [12 (link)], and subjected to antigen retrieval by microwave (320 W, 11 min). The cyclooxygenase-2 was stained according to the commercial kit (Solarbio, Shanghai, China).
Slices were deparaffinized and rehydrated with standard methods, then processed with Hematoxylin staining for 5–10 min, differentiated with 0.5% hydrochloric acid ethanol for 30 s, and stained with 0.5% Eosin for 10 s, following by rinsing with tap water for 25 min. Finally, the slices were dehydrated with standard protocol and sealed.
For immunohistochemistry assay, slices were deparaffinized and rehydrated by standard methods [12 (link)], and subjected to antigen retrieval by microwave (320 W, 11 min). The cyclooxygenase-2 was stained according to the commercial kit (Solarbio, Shanghai, China).
3
Colon Tissue Harvesting and Analysis
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Mice were sacrificed 21 days after the first administration of DSS. Mice were anesthetized by injection of Zoletil 50® (06516, 10 mg/kg, Vibac Laboratories, Carros, France) intraperitoneally. Laparotomy was performed, and distal colon segments from the same defined area were collected. To analyze colon length and morphological changes, segments of the colon were photographed and the colon was weighted. The collected colon tissues were immediately frozen at −80 °C, and some were used for Western blotting. The remaining colon tissues were used for histological staining after fixation with 4% paraformaldehyde solution in 100 mM phosphate buffer (pH 7.4) for 24 h. Colon tissues were dehydrated in various ethanol concentrations, embedded in paraffin, and sectioned at 5 μm in thickness by a paraffin microtome (Thermo Fisher Scientific, Somerset, NJ, USA). Colon tissue sections were then mounted on gelatin-coated slides and dried overnight in an oven at 37 °C.
4
Hepatic Injury Analysis in Mice
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Blood sampling was performed first, and tissue preparation was conducted in the same manner as described above [12 (link),39 (link)]. Mice were weighed before sacrifice and CCl4 was first injected and mice were sacrificed 7 days later. After anesthetizing the mice with Zoletil 50® (10 mg/kg; Vibac Laboratories, Carros, France), blood was drawn by cardiac puncture and then maintained at room temperature for 1 h. Blood was centrifuged at 3000 rpm for 20 min to obtain serum.
After blood sampling, physiological saline was perfused through the portal vein, and livers were collected. The wet weight of the mouse liver tissue was measured. The liver index was calculated using body weight and liver wet weight, and the formula is as follows. Liver index = liver weight/body weight ×100 (%). The liver was then fixed in 4% paraformaldehyde, dehydrated with graded ethanol, treated with xylene, infiltrated and embedded in paraffin. Thereafter, coronal sections with a thickness of 5 μm were prepared using a paraffin microtome (Thermo Fisher Scientific, New Jersey, NJ, USA), the sections were mounted on coated slide, and then the slides were dried on a hot plate at 37 °C overnight. An average of 6 slice sections from each liver were collected and used in the next experiment.
After blood sampling, physiological saline was perfused through the portal vein, and livers were collected. The wet weight of the mouse liver tissue was measured. The liver index was calculated using body weight and liver wet weight, and the formula is as follows. Liver index = liver weight/body weight ×100 (%). The liver was then fixed in 4% paraformaldehyde, dehydrated with graded ethanol, treated with xylene, infiltrated and embedded in paraffin. Thereafter, coronal sections with a thickness of 5 μm were prepared using a paraffin microtome (Thermo Fisher Scientific, New Jersey, NJ, USA), the sections were mounted on coated slide, and then the slides were dried on a hot plate at 37 °C overnight. An average of 6 slice sections from each liver were collected and used in the next experiment.
5
Histological Examination of Colon Tissue
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