The samples of cecum fixed in 4% paraformaldehyde solution were embedded in paraffin to generate 5-μm transverse sections and stained with Alcian blue (Servicebio Technology Co., Ltd., Wuhan, China) for microscopic examination. The sections were pictured by a Sony Alpha6000 APS camera. Then, villus height (VH), crypt depth (CD), muscle layer thickness (MLT), and the number of goblet cells per villus of cecum were determined by Image Pro-Plus 6.0 software (Media Cybernetics, Bethesda, MD, United States).
Alcian blue
Manufactured by Wuhan Servicebio Technology
Sourced in China
Alcian blue is a histological stain used to detect the presence of acidic polysaccharides, such as glycosaminoglycans, in biological samples. It binds to these molecules, producing a blue color that can be observed under a microscope. Alcian blue is commonly used in various fields of research, including histology, cytology, and tissue engineering, to identify and visualize specific components within cells and tissues.
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Lab products found in correlation
Image pro plus 6, by Media Cybernetics (1 mentions)
Alpha6000 aps camera, by Sony (1 mentions)
Bx53 fluorescence microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ivision mac scientific imaging processing software, by Olympus (1 mentions)
G1053, by Wuhan Servicebio Technology (1 mentions)
Safranin o, by Wuhan Servicebio Technology (1 mentions)
Ventana dp 200, by Roche (1 mentions)
Dissecting microscope, by Leica (1 mentions)
Alizarin red, by Yeasen (1 mentions)
Eclipse ci, by Nikon (1 mentions)
Picrosirius red, by Wuhan Servicebio Technology (1 mentions)
Masson trichrome, by Wuhan Servicebio Technology (1 mentions)
Periodic acid schiff, by Wuhan Servicebio Technology (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Alizarin red s, by Wuhan Servicebio Technology (1 mentions)
Oil red o staining, by Wuhan Servicebio Technology (1 mentions)
Toluidine blue, by Wuhan Servicebio Technology (1 mentions)
Citrate buffer, by Wuhan Servicebio Technology (1 mentions)
9 protocols using alcian blue
1
Histomorphometric Analysis of Cecum
2
Histological Analysis of Olfactory Mucosa in Rainbow Trout
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The OM tissue of rainbow trout was cut accurately, and the eyeball and muscle tissue beneath the mucosal layer removed carefully using anatomical scissors. Then, the tissues were processed for routine histology as described previously (Kong et al. 2019 (link); Yu et al. 2018 (link), 2019 (link)). Briefly, all OM tissue samples were fixed in 4% neutral buffered formalin (1:10) overnight at 4 °C and then were dehydrated through an alcohol gradient, mounted in paraffin, and cut into 5 μm slices. Sections were stained with hematoxylin–eosin (H&E, Biosharp, China) and alcian blue (A&B, Servicebio, China), which was used to check that most of the goblet cells of the OM were blue stained (acidophilic). Quantification was performed by three different researchers as in the previous study (Zhang et al. 2018 (link)). Immunofluorescence staining was employed to observe the location and distribution of Ich. The OM sections were incubated with anti-Ich polyclonal Ab (mouse IgG1, 1 μg ml−1) or isotype control mouse IgG1 overnight at 4 °C. Nuclei were stained with DAPI (Invitrogen, USA) before mounting. Images were acquired and analyzed using the Olympus BX53 fluorescence microscope and the iVision-Mac scientific imaging processing software (Olympus).
3
Cartilage Matrix Mineralization Analysis
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Chondrocytes cocultured with or without BMSCs were first fixed with 4% paraformaldehyde for 30-45 min and then stained with Alcian blue (G1027, Servicebio, Wuhan, China) and safranin O (G1053, Servicebio, Wuhan, China) to detect the extent of matrix mineralization. Quantification of Alcian blue staining was performed by measuring the absorbance at 620 nm after dissolving the stained micromass with 6 M guanidine hydrochloride solution [33 (link)]. safranin O staining was washed out in isopropanol and incubated for 30 min with gentle agitation. Each sample was quantified as optical density in a microplate reader at 540 nm [34 (link)]. For tissue Masson staining and safranin O staining, the fixed samples were embedded for sectioning and staining.
4
Histological Analysis of Colonic Inflammation
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For morphological measurements, proximal colon tissues were consecutively sectioned at 5 μm thickness and stained with hematoxylin and eosin (H&E). Scanning images were evaluated using the Image Viewer v3.2 software. The extent of inflammatory infiltration, histopathological changes in crypt structure, ulceration and crypt loss, ulcer, and presence of edema were measured. Histological scores were determined as described previously [18 (link)]. To count colonic goblet cells, Carnoy’s fixed colonic tissues were stained in Alcian blue following manufacturer’s instructions (Servicebio, Wuhan, China). Briefly, sections were stained with Alcian blue solution for 10 to 15 min, and then were rinsed with distilled water. Slides were dehydrated with absolute ethyl alcohol and xylene followed by image acquisition on slide scanner VENTANA DP 200 (Roche, USA). Goblet cell counts were calculated as the average of the number of goblet cells found in 10 randomly chosen intact crypts per mouse.
5
Skeletal Staining Protocol for Zebrafish Larvae
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For a fixed sample, all specimens (8 dpf larvae) were anesthetized using tricaine and then fixed in 4% paraformaldehyde for 1 hour at room temperature. They were subsequently washed and dehydrated with 50% ethanol for 10 minutes at room temperature. To the larvae, 0.5% Alizarin Red (Yeasen, 60504ES25, Shanghai, China) or 0.1% Alcian Blue staining (Servicebio, G1027, Wuhan, China) Solution was added, and staining was done overnight at room temperature. After elution of the staining solution by gradient ethanol, a final concentration of 1.5% H202 and 1% KOH bleach solution was added for bleaching. Final decolorization was performed overnight with 20% glycerol and 0.25% KOH solution, followed by 50% glycerol and 0.25% KOH solution, respectively. The larvae were observed under a Leica dissecting microscope.
6
Histological Evaluation of Tissue Samples
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Native and decellularized samples were fixed in 4% paraformaldehyde, dehydrated in graded alcohol series, embedded in paraffin, and sectioned into 5-μm sections. The glass slides were stained with hematoxylin and eosin (H&E, Servicebio, Wuhan, China), Masson trichrome, periodic acid–Schiff, Alcian blue, and picrosirius red (PSR) according to the manufacturer’s instructions. All reagents were purchased from Servicebio. The sections were imaged on a light microscope (CX43, Olympus, Tokyo, Japan). PSR-stained slides were assessed by phase-contrast microscopy (Eclipse Ci, Nikon, Tokyo, Japan) and analyzed by the CT-FIRE program.
7
Differentiation Analysis of Tissue-Specific Progenitor Cells
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The TSPCs were induced for osteogenic, adipogenic, and chondrogenic differentiation analysis using previously established methods.22 (link) After induction, the cells were stained with Alizarin Red S (Servicebio) for osteogenesis analysis. The cell pellets, stained with Alcian blue (Servicebio) solution and type II collagen (1:400, Sigma, CP18) antibody, were used for chondrogenesis analysis. Oil Red O staining (Servicebio) was used for adipogenesis analysis. To quantify mineralization of Alizarin Red S staining, calcium deposits were incubated with 10% cetylpyridinium chloride (Sigma), and their concentration was measured at 570 nm absorbance. Quantification of the aggrecan-positive area and Oil Red O-positive area was performed using Image J software.
8
Colonic Mucus Layer Measurement
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Colon was removed, fixed with Carnoy’s fixative, embedded in paraffin, and sliced into 5 µM sections. The paraffin sections were dewaxed, dehydrated, and stained with alcian blue (Servicebio, G1049). The thickness of the colonic mucus layer was measured on 10 random visual fields per section using ImageJ software (NIH, USA).
9
Histological Analysis of Decalcified Rat Knee
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The decalcified rat knee tissues were collected, dehydrated, and embedded in paraffin. They were cut into 4 μm-thick sections and stained with hematoxylin and eosin (HE) (Servicebio, China), safranin O-fast green (Servicebio, China), alcian blue (Servicebio, China), and toluidine blue (Servicebio, China). Additionally, immunohistochemical staining was performed using specific antibodies against ACAN, COL2A1, MMP3, and MMP13. The heart, liver, spleen, lung, and kidney of the rats were stained only with HE.
For the immunofluorescence staining, the sections were dewaxed, rehydrated, repaired with citrate buffer (Servicebio, China), blocked with 3% H2O2 for 30 min, and then blocked with 5% bovine serum albumin (Servicebio, China) for 1 h. Subsequently, the sections were incubated with the specific antibody against COL2A1 at 4°C overnight and incubated with the corresponding secondary antibody at room temperature in the dark for 30 min on the next day. The nuclei were stained with destination access point identifier dye (Servicebio, China), and the sections were observed under a fluorescence microscope.
For the immunofluorescence staining, the sections were dewaxed, rehydrated, repaired with citrate buffer (Servicebio, China), blocked with 3% H2O2 for 30 min, and then blocked with 5% bovine serum albumin (Servicebio, China) for 1 h. Subsequently, the sections were incubated with the specific antibody against COL2A1 at 4°C overnight and incubated with the corresponding secondary antibody at room temperature in the dark for 30 min on the next day. The nuclei were stained with destination access point identifier dye (Servicebio, China), and the sections were observed under a fluorescence microscope.
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