The left ventricular anterior wall tissue of each group of rats was collected and immersed in 4% paraformaldehyde fixative for 24 h. The tissue was then dehydrated in gradient alcohol and placed in paraffin to make a paraffin mass. Then, we used a microtome (RM2235, Leica, Wetzlar, Germany) to make paraffin sections. We dewaxed and hydrated paraffin sections in xylene and gradient alcohol. The sections were then rinsed in running water for 3 min. The sections were placed in hematoxylin stain (Beyotime, Shanghai, China) for 1 min. After rinsing under running water, we placed the sections in hydrochloric acid alcohol (Beyotime, Shanghai, China) for another 3 s. The sections were then rinsed with running water, placed in eosin staining solution (Beyotime, Shanghai, China) for 1 min, and dehydrated to seal the sections.
Hematoxylin stain
Manufactured by Beyotime
Sourced in China
Hematoxylin stain is a commonly used laboratory reagent in histological and cytological applications. It is a natural dye derived from the heartwood of the Logwood tree (Haematoxylum campechianum). The stain selectively binds to acidic structures within cells, such as the nuclei, and imparts a blue-purple color. It is often used in combination with other counterstains to provide contrast and aid in the visualization of cellular and tissue structures during microscopic examination.
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5 protocols using hematoxylin stain
1
Histological Analysis of Rat Cardiac Tissue
2
Histological Analysis of Thyroid Tissue
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The pathological tissues were obtained by surgery, and fixed with 10% formaldehyde and then embedded in paraffin and sliced at a thickness of 4 µm. For hematoxylin and eosin (H&E) staining, paraffin-embedded thyroid tissue sections were deparaffinized with xylene for 5 min (this process was repeated once), 100% ethanol for 5 min, 90% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min, and washed with distilled water for 2 min. The sections (4-µm-thick) were stained with hematoxylin stain (Beyotime Institute of Biotechnology, Inc.) for 5 min and washed with distilled water for 10 min at room temperature. The sections were differentiated with acid alcohol slow differentiation solution (Beyotime Institute of Biotechnology, Inc.) for 30 sec and soaked in distilled water for 15 min. The sections were stained with eosin stain (Beyotime Institute of Biotechnology, Inc.) for 2 min at room temperature. Slides were viewed with a Nikon Eclipse 80i microscope equipped with a digital camera (DS-Ri1; Nikon Corp.).
3
Hematoxylin-Eosin Staining of MOVAS Cells
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Exactly 1 mL of cell suspension with an MOVAS cell concentration of 1.5×105 cells/mL was seeded per well in 12-well plates. The experimental grouping was similar to that in the section on CCK-8. After incubation for 24 h, the supernatant was removed, and the cells were washed twice with PBS. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then washed three times with PBS. After fixation, the cells were stained with hematoxylin stain (Shanghai Beyotime Bio-Tech Co., Ltd.) and incubated for 15 min. They were then washed three times with distilled water for 2 min to remove excess stains. Thereafter, the cells were stained with eosin staining solution for 5 min. The cells were washed three times with distilled water for 2 min to remove excess eosin. After treatment, the cells were observed under a microscope (CKX41; Olympus, Tokyo, Japan).
4
Histological Processing of Rat Myocardial Tissue
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After collecting the rat heart, we used PBS to wash away excess blood from the heart. Then, we used 4% paraformaldehyde to fix myocardial tissue for 24 h. The fixed tissue can be stored in PBS for a long time. We put myocardial tissue into gradient alcohol in order to dehydrate. Then, we put myocardial tissue in xylene and paraffin solution to make paraffin blocks. The microtome is used to make paraffin sections with the thickness of 5 μm. We put paraffin sections in a 37°C incubator for 3 d. Then, we put the paraffin sections in xylene solution and gradient alcohol sequentially. After washing the sections with running water, we stained the cell nucleus with hematoxylin stain (Beyotime, Shanghai, China). Then, we put the sections in 1% hydrochloric acid alcohol for 3 s and quickly rinsed the sections with running water. Then, we put the sections in eosin stain (Beyotime, Shanghai, China) and gradient alcohol in turn for 3 min each time. Finally, we use neutral gum for mounting and observe the staining results using an optical microscope.
5
Hematoxylin-Eosin Cell Staining
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After repair, cells were fixed with 4% paraformaldehyde and then stained with hematoxylin stain (Beyotime, Shanghai, China) for 15 min. After wash, cells were further dyed with eosin (Beyotime, Shanghai, China) for 5 min. The cells in the 12-well plate were observed under an optical microscope (OLYMPUS, CKX41, Japan).
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