The heads of the mice were fixed in neutral 10% formalin fixative (PH7.4, Solarbio, Beijing, China), successively dehydrated by 80%, 90%, 95% and 100% ethyl alcohol (Sigma, California, US), and made transparent by xylenes I & II (Sigma, CA, USA). Next, the tissues were paraffin-embedded (Chemical Book, Beijing, China), and cut into 5-μm-thick sections, washed with xylenes and ethyl alcohol for debenzolization and rehydration, and then stained with a hematoxylin and eosin (H&E) staining kit (ab245880, Abcam, USA). Next, dehydration and transparentizing of the tissue slices were conducted again using ethyl alcohol and xylenes. Finally, the samples were sealed in neutral balsam (Macklin, Shanghai, China). The epithelial damages were observed under a microscope with 1000× magnification.
Neutral balsam
Manufactured by Maclin Biochemical Technology
Sourced in China
Neutral balsam is a transparent, viscous liquid used as a mounting medium in microscopy. It has a refractive index similar to glass, allowing for clear visualization of specimen details. Neutral balsam is a common choice for preparing and preserving samples for long-term storage and observation.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Ab245880, by Abcam (1 mentions)
Ethyl alcohol, by Merck Group (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Optical microscope, by Thermo Fisher Scientific (1 mentions)
Haematoxylin h3136, by Merck Group (1 mentions)
N861409, by Maclin Biochemical Technology (1 mentions)
Primotech, by Zeiss (1 mentions)
4 protocols using neutral balsam
1
Histological Analysis of Mouse Tissue
2
TUNEL Assay for Apoptosis Detection
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TUNEL assays are carried out using a commercial kit (KeyGEN, KGA703). According to the instructions provided by the manufacturer, freezing sections were fixed in 4% paraformaldehyde fix solution at room temperature (15-25°C) for 20-30 minutes and rinsed in 1 × PBS three times for 15 minutes. Then, sections were treated with 100 μL proteinase K for 30 minutes at 37°C and rinsed in 1 × PBS three times for 15 minutes again. After being immersed in 3% H2O2 sealing liquid for 10 minutes and rinsed, the slides were added with 100 μL DNase I reaction liquid containing 2000-3000 U, 40-60 μL DNase I (50 U/μL), and 60-40 μL DNase I buffer and rinsed. Later, TdT enzyme reaction solution, streptavidin-HRP working fluid, and DAB working fluid were added 50 μL after each time the slides were rinsed in 1 × PBS three times for 15 minutes and drained with blotting paper. Dyed in hematoxylin stain for 30 seconds to 5minutes and washed with distilled water, the slides were put in methanol hydrochloride solution for differentiation for 5 seconds and washed with distilled water again, followed by 70%, 85%, 95%, and absolute ethyl alcohol each for 5-minute rinse and xylene twice for ten minutes. After being dried, the samples were added with neutral balsam (Macklin, 822941-100 g), covered with glass slides, photographed, and observed under an optical microscope.
3
Histological Analysis of ALI Murine Lungs
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Lung tissues of ALI mice were fixed in 10% paraformaldehyde (16005, Sigma-Aldrich, USA) for 24 h, dehydrated by gradient alcohol, transparentized by xylene (95682, Sigma-Aldrich, USA), embedded in paraffin (1496904, Sigma-Aldrich, USA) and cut into 5-μm thick sections. Then, after being dewaxed by xylene and rehydrated by gradient alcohol, the sections were stained with Hematoxylin (H3136, Sigma-Aldrich, USA) for 12 min and eosin for 5 min. After staining, the sections were sealed with neutral balsam (N861409, Macklin, Shanghai, China) and dried at 37℃ for 4 hours. The histopathological changes of the lung tissues were observed by an optical microscope (13132071, fisherscientific, Massachusetts, USA) under 100× magnification.
4
Histopathological Analysis of Rat Liver and Intestine
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After rat liver and intestinal mucosa were fixed in 4% paraformaldehyde (16005, Sigma-Aldrich, St. Louis, MO) for 24 h, they were dehydrated by gradient alcohol, transparentized by xylene (95682, Sigma-Aldrich, St. Louis, MO), and embedded in paraffin (1496904, Sigma-Aldrich, St. Louis, MO). Subsequently, the paraffinized tissues were cut into 5 μm thick sections, dewaxed by xylene and rehydrated by gradient alcohol. Then, the sections were stained with haematoxylin (H3136, Sigma-Aldrich, St. Louis, MO) for 12 min. After being differentiated by hydrochloric alcohol, the sections were stained with eosin (E4009, Sigma-Aldrich, St. Louis, MO) for 5 min. Thereafter, the stained sections were sealed by neutral balsam (N861409, Macklin, Shanghai, China) and dried at 37 °C for 4 h. Afterwards, the histopathological changes in the liver tissues and intestinal mucosa tissues were observed by an optical microscope (ZEISS Primotech, Carl Zeiss, Oberkochen, Germany) under magnifications of ×200 and ×40, respectively.
The morphological changes in liver were determined by NAFLD activity score, which includes histological features and has been defined as unweighted sum of scores for steatosis (0–3), lobular inflammation (0–3) and ballooning (0–2).
The morphological changes in liver were determined by NAFLD activity score, which includes histological features and has been defined as unweighted sum of scores for steatosis (0–3), lobular inflammation (0–3) and ballooning (0–2).
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