We used paraffin to embed the lung tissue and cut the tissue into thin slices. Next, xylene was used for the dewaxing of these tissues. After that, these tissues were incubated with H2O2 and stewed in the citrate buffer solution to expose the antigen sites. Then, these tissues were blocked with BSA (Beyotime, China) and incubated with the primary antibody against LC-3 (ab48394, Abcam) at 4°C overnight. On the second day, the tissues were incubated with the secondary antibody (Goat Anti-Rabbit IgG H&L, ab205718, Abcam) at room temperature for 2 hours. Then, these tissues were stained with the hematoxylin solution (Beyotime, China). Finally, the expression of LC-3 in these tissues was observed under the microscope (Olympus, Japan).
Hematoxylin solution
Manufactured by Beyotime
Sourced in China
Hematoxylin solution is a laboratory reagent used for staining biological samples. It is a natural dye derived from the Logwood tree and is commonly used in histology and cytology to stain cell nuclei, enabling the visualization of cellular structures under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Eosin solution, by Beyotime (6 mentions)
Streptavidin horseradish peroxidaseconjugated antibody, by Abcam (3 mentions)
3 3 diaminobenzidine tetrahydrochloride dab solution, by Yeasen (3 mentions)
Primary antibody against bcl 2, by Abcam (3 mentions)
Ab48394, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Goat anti rabbit igg h l ab205718, by Abcam (1 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
Dmi4000b, by Leica (1 mentions)
Bx53f inverted microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Masson trichrome staining kit, by Beyotime (1 mentions)
Hydrochloric acid alcohol, by Beyotime (1 mentions)
Dp71 ccd, by Olympus (1 mentions)
Neutral balsam, by Beyotime (1 mentions)
Xylene, by Beyotime (1 mentions)
Gradient ethanol, by Sinopharm (1 mentions)
Xylene, by Sinopharm (1 mentions)
14 protocols using hematoxylin solution
1
Immunohistochemical Analysis of LC-3 Expression
2
Histopathological Analysis of Ocular Inflammation
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For sagittal sections extracted from the eyes on day 1 PII of LPS or PBS, hematoxylin-eosin (HE)staining was conducted according to the manufacturer’s instructions. Briefly, sections were stained with hematoxylin solution (Beyotime, C0105S) for 5 min followed by 5 dips in 1% acid ethanol (1% HCl in 70% ethanol) and then rinsed in distilled water for 5 min. The sections were stained with eosin solution (Beyotime) for 3 min before rinsing in distilled water for 5min, followed by dehydration with a gradient solution of ethanol (70% ethanol for 10 s, 80% ethanol for 10 s, 90% ethanol for 10 s, and absolute ethanol for 10 s) and then submersion in xylene. The mounted slides were examined and photographed using a light microscope (Olympus) at 10X magnification. The inflammatory cells infiltrating into the retina, vitreous cavity and ONH were evaluated.
3
Histological Staining and Microscopy
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After the paraffin sections were dewaxed and watered with xylene and gradient alcohol, the nuclei were stained with hematoxylin solution (Beyotime, Shanghai, China), the eosin solution was used for staining cytoplasm (Beyotime, Shanghai, China), and the slides were mounted with neutral resin. Pictures were taken with an Olympus BX51 fluorescence microscope (Olympus, Japan).
4
Transwell Assay for Cancer Cell Migration
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DU-145 cells were seeded into the insert of Transwell (Corning, Tewksbury, MA) at a density of 1 × 105 cells/well, then cultured in serum-free culture media. Fucoidan (500 μg/mL) or vehicle (PBS) was added to the lower reservoirs. Cells were subsequently allowed to migrate across a collagen I-coated polycarbonate filter for 12 h at 37 °C. Non-migrated cells were removed from the top side of the filter by scraping. Migrated cells on the bottom side of the filter were subsequently fixed with 4% paraformaldehyde for 30 min and stained by hematoxylin solution (Beyotime, Shanghai, China) for 5 min. Cells in five random fields of each migration well were counted to determine the average number of migrated cells.
5
Characterizing Bioprinted 3D Structures
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We utilized HE and immunofluorescence staining to examine the morphology and stability of the induced MTs in the bioprinted 3D structure. After the bioprinting process was finished, the 3D structure was embedded in optimal cutting temperature compound (OCT, Sakura) and subjected to frozen sectioning for HE and immunofluorescence staining. For HE staining, frozen sections were washed briefly in distilled water, stained in hematoxylin solution (Beyotime Biotechnology, China) for 5 min, washed in running tap water for 1 min, differentiated in 1% acid alcohol for 6 s, washed in running tap water again for 1 min, blued in 0.2% ammonia water, washed in running tap water for 1 min, counterstained in eosin solution (Beyotime Biotechnology, China) for 5 min, dehydrated in alcohol (75% × 2 for 2 s each, 80% × 2 for 2 s each, 95% × 2 for 1 min each, 100% × 2 for 1 min each), and cleared in 2 changes of xylene for 3 min each. For immunofluorescence staining, we followed the protocol above. Samples were photographed and recorded using a LEICA DMI 4000B digital microscope camera system (Leica, Heidelberg, Germany).
6
Histological Sample Preparation Protocol
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The samples were first immobilized in 4% paraformaldehyde (Solarbio, Beijing, China) for 24 h, then immersed in 70%, 80%, 90%, 95%, and 100% ethanol solutions for 30 min to dehydrate the samples. They were then placed in xylene for 2 h to make the samples transparent and embedded in paraffin wax for 3 h. The embedded samples were sectioned into 5 μm pieces and immersed in xylene for 20 min to dewax the samples. The sections were then immersed in a series of ethanol solutions from high to low concentrations and finally in distilled water. The sections were stained with a hematoxylin solution (Beyotime, Haimen, China) for 4 min, fractionated in hydrochloric acid and ethanol for 3 s each, rinsed in running water for 1 h, immersed in distilled water for 10 min, and dehydrated in 70% and 90% ethanol solutions for 10 min each, followed by staining with the eosin staining solution for 3 min. The stained sections were dehydrated by immersion in an ethanol solution and then immersed in xylene to make the sections transparent, and they were finally sealed and stained with gum. The sections were sealed with resin, observed under a BX53F inverted microscope (Olympus, Tokyo, Japan), and photographed.
7
Surgical HCC Resection Protocol
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The experimental protocol was approved by the Institutional Ethics Review Board of Daping Hospital, the Third Military Medical University. Written informed consent was obtained from all participants. A total of 10 patients with HCC were recruited randomly at inpatient service of the Department of Hepatobiliary Surgery, the Third Military Medical University between 2013 and 2014. Patients underwent surgical HCC resection; HCC and corresponding non-tumor liver tissues were collected. All specimens were snap-frozen in liquid nitrogen or stored at −80°C. The crystal HCC and non-tumor tissue sections (4 µm) were stained with H&E: Sections were mounted on glass slides, fixed with fixative (4% paraformaldehyde) for 20 min at room temperature, air-dried for 15 min, stained in hematoxylin solution (Beyotime Institute of Biotechnology, Haimen, China) for 2 min at 42°C, then washed in running distilled water for 10 min. Subsequently, sections were stained in 0.5% eosin solution (Beyotime Institute of Biotechnology) for 1 min at 42°C, and then washed again, dehydrated, and mounted at room temperature to ensure homogenous cell population of tissues. Individual patients were excluded if they received any chemotherapy and radiotherapy. Among those 10 patients, 7 were male and 3 were female. The age ranged from 35–72 years; 8 patients were serum positive for hepatitis B surface antigen.
8
Histological Analysis of Mouse Lumbar Spine
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The fixed lumbar spines of mice were decalcified in 23% ethylenediaminetetraacetic acid at 4 °C for 5–7 days and embedded in paraffin. Bone sections (4 μm) were deparaffinized, and washed with water for 3 min. Then hematoxylin solution (Beyotime, Shanghai, China) was applied for 10 min, washed with water, hydrochloric acid ethanol for 30 s, counterstained with eosin solution, and dehydrated. All the analyses were conducted in a blinded fashion.
9
Immunohistochemical Analysis of BCL2 in CRC
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The tumor samples from CRC patients were fixed in 10% formaldehyde, embedded in paraffin and then sectioned into slices. For IHC assay, tumor slices were firstly deparaffinized and rehydrated. After washing, slices were treated with H2O2 to reduce the endogenous peroxidase activities. Slices were then incubated with primary antibody against BCL2 (1:50, Abcam, Cambridge, UK) overnight at 4 °C. With three times washing in PBS, the slides were incubated with secondary streptavidin–horseradish peroxidase-conjugated antibody (1:3000, Abcam, Cambridge, UK) for 1 h, and reacted with 3,3-diaminobenzidine tetrahydrochloride (DAB) solution (Yeasen Biotech, Shanghai, China) for 5 min. Finally, slides were counterstained with hematoxylin solution (Beyotime Biotechnology, Shanghai, CHN) for 1 min, dehydrated and mounted with neutral gum.
10
Histological Analysis of Rat Myocardial Tissue
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Rat hearts were collected, and myocardial tissue was washed with normal saline. Then, we used 4% paraformaldehyde to fix myocardial tissue. We used gradient alcohol for dehydration and embed myocardial tissue in paraffin. Then, we used a microtome to make paraffin blocks into paraffin sections. Before hematoxylin-eosin (HE) staining, we put paraffin slices in xylene and gradient alcohol for dewaxing and hydration. Then, we stained the cell nucleus with hematoxylin solution (Beyotime, Shanghai, China) for 1 minute. After rinsing slices with running water, we used 1% hydrochloric acid alcohol for differentiation. Then, we used eosin solution (Beyotime, Shanghai, China) to stain the cytoplasm. Finally, we used neutral gum for sealing. In addition, we used Masson trichrome staining kit (Beyotime, Shanghai, China) for staining according to the manufacturer's instructions. Collagen fibers appear blue and muscles appear red.
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