To assess the degree of myelination/demyelination, staining with the Luxol Fast Blue (LFB) stain kit (Abcam, Waltham, MA 02453, USA # ab150675) was performed as briefly described below. Sections were deparaffinized and incubated in LFB solution at 56 °C O/N, then washed in 95% alcohol. Subsequently, the sections were incubated in lithium carbonate solution and 70% ethyl alcohol and finally counterstained in the cresyl violet solution. After dehydration, the sections were assembled with Eukitt (Bio-Optica, Milan, Italy) and observed by a light microscopy Eclipse Ci-L microscope. The slides were analyzed by a pathologist blinded to the treatment groups. Images were taken focusing on the perilesional area of the SCI and shown at 20× and 40× magnification.
Luxol fast blue stain kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The Luxol Fast Blue Stain Kit is a laboratory tool used for the histological staining of myelin in tissue samples. The kit provides the necessary reagents and instructions to perform this specific staining technique.
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Lab products found in correlation
Xylene, by Samchun (2 mentions)
Eukitt, by Bio-Optica (1 mentions)
Kinase assay buffer, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Vybrant mtt cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
Mini edta free protease inhibitor, by Roche (1 mentions)
Endofectin max transfection reagent, by GeneCopoeia (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Zen 3.1 blue edition, by Zeiss (1 mentions)
Hrp dab abc detection ihc kit, by Abcam (1 mentions)
9 protocols using luxol fast blue stain kit
1
Luxol Fast Blue Staining for Myelination
2
Characterization and Validation of GMF-β Compound
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GMFBI.1 compound was characterized by NMR and HPLC with >95% purity ( Supplementary Fig. S15 ) was purchased from Specs (Netherlands), custom made rabbit anti-phospho Ser83 specific antibodies to GMF-β conserved between mouse, rat and human sequence 76–90 (QHDDGRVS(p)YPLCFIF; Product YZ6909 is from YenZyme, USA), MOG 35–55 (MEVGWYRSPFSRVVHLYRNGK) peptide, hGMF-β construct and EndofectinTM Max transfection reagent from GeneCopoeia (USA), Pertussis toxin from List biological (USA), Mtb H37RA from Difco (USA). GMF-β from Origene (USA). Haematoxylin, Eosin, Chemiluminescent solution Luminata Forte, PKA, Adenosine triphosphate (ATP), LPS [E. coli 0111:B4], complete Freund’s adjuvant and Toluidine blue stain were from Sigma Aldrich-Millipore (USA), DMEM-F12 from Gibco (USA), Fetal Bovine Serum (FBS), Penicillin/Streptomycin, Trypsin (0.01%) were from Invitrogen (USA), protease inhibitor cocktail was from cOmplete, Mini EDTA-free protease inhibitor tablets Roche (Germany), antibodies against GMF-β, NF-κB p65, p38MAPK and Luxol fast blue (LFB) stain kit were from Abcam (UK), β-actin from Santacruz (USA), AST and ALT kit from Aspen (India), mouse Cytokines Multi-Analyte ELISA Array Kit- Qiagen (Germany), Vybrant-MTT cell proliferation assay kit and kinase assay buffer were from Thermo Fisher Scientific (USA).
3
Histological Analysis of Spinal Cord and Liver
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The lumbar sections of the spinal cord (15 μm) were prepared and stained using Luxol Fast Blue (LFB) Stain Kit (ab1506751; Abcam, Cambrdge, UK) according to the manual. The prepared liver sections were stained using the hematoxylin & eosin (H&E) method: the sections were first soaked in 0.4% HCl (in EtOH) for 15 s, washed once, and then stained with eosin for 3 min. The LFB and H&E slides were then imaged to examine the pathological changes of tissues.
4
Quantitative Demyelination Analysis in Mice
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To evaluate demyelination in the mouse brain and spinal cord, fixed tissues were stained using the Luxol Fast Blue Stain Kit (Abcam), as described in the manufacturer’s protocol. Mice were sacrificed and assessed at the peak EAE stage, approximately 17–19 days post immunization. The sections were incubated with 0.1% Luxol fast blue solution at 24℃ overnight and differentiated in 0.05% lithium carbonate solution at 24℃. When differentiation was completed, the sections were counterstained with 0.1% cresyl echt violet solution for 3 min at 24℃. Analysis of demyelination was conducted in both the brain corpus callosum region and the thoracic to lumbar region of spinal cord. The demyelinated regions in the white matter were quantified using ImageJ (NIH, USA) [37 (link), 38 (link)].
5
Evaluating Spinal Cord Demyelination
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LFB staining was performed on the spinal cord tissue to evaluate the demyelination using a Luxol Fast Blue Stain Kit (Abcam, Cambridge, UK, #ab150675) at 28 DPI. The lesion sections of the spinal cord were deparaffinized in Xylene (Samchun Chemical, Pyungtaek, Korea) and dehydrated in ethanol solution that was graded and incubated in LFB solution at room temperature overnight. The samples were differentiated in a lithium carbonate solution. Scanning was conducted using an Olympus C-mount camera adapter (U-TVO.63XC, Tokyo, Japan).
6
Evaluating Spinal Cord Myelination after Injury
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To evaluate the extent of myelination, LFB staining was performed on spinal cord tissue samples of dpi 1 and 20 using Luxol Fast Blue Stain Kit (Abcam, Cambridge, UK, #ab150675). The spinal cord tissue sections were deparaffinized in xylene (Samchun chemicals, Seoul, Korea) and dehydrated in graded ethanol solutions and incubated in LFB solution at room temperature for 24 h. The sections were differentiated in the lithium carbonate solution followed by 70% ethyl alcohol and were then counterstained with cresyl violet. Lastly, the sections were dehydrated in 100% alcohol, cleared in xylene, and mounted using mounting solution (Canada balsam, Junsei, Tokyo, Japan, #23255-1210). Scanning was done with OLYMPUS C-mount camera adapter (U-TVO.63XC, Tokyo, Japan). The total area of cavitation and LFB-positive myelinated areas at the injury epicenter (0 mm), as well as 1–2 mm rostral and caudal areas from the epicenter were measured using the Zeiss analysis program (Zen 3.1 Blue edition, Zeiss, Thornwood, NY, USA).
7
Dual Staining of Myelin and Nissl
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Luxol Fast Blue Stain Kit (Abcam) is used to stain myelin/myelinated axons and Nissl bodies. Firstly, deparaffinize the sections and hydrate in water. And then incubate in Luxol Fast Blue solution for 24 h at room temperature. After that, rinse the sections in water, differentiate by dipping in lithium carbonate solution, and differentiate further by dipping in alcohol reagent. And this step would be repeated three times. Lastly, incubate the sections in Cresyl Echt Violet for 2-5 min to clear and mount for consecutive analysis.
8
Luxol Fast Blue Staining for CNS Demyelination
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LFB is widely used to detect demyelination in the CNS. Coronal Sects. (20 μm) were stained with Luxol Fast Blue Stain Kit according to the manufacturer’s instructions (Abcam, USA) and conducted as described previously [20 (link)]. The severity of the white matter lesions was graded as described [21 (link)].
9
Spinal Cord Histological Analysis
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Spinal cords were fixed and processed in paraffin blocks for histological analysis. Tissues were sectioned horizontally at 5 μm. Sectioned samples on the slides were deparaffinized and hydrated by incubating slides in xylene for 15 min, 3 times, and 100% and 95% ethanol for 5 min, 2 times each. Then, an antigen unmasking procedure was conducted by boiling in 10 mM sodium citrated buffer (pH 6) for 10 min. Mouse-/rabbit-specific HRP/DAB (ABC) detection IHC kit (Abcam) was used following the manufacturer’s recommended protocol to develop slides by the DAB substrate. Anti-mouse CD138 and anti-rabbit CD4 polyclonal antibodies for use as primary antibodies were purchased from Thermo Fisher Scientific. Infiltrated cells and myelinated area were measured by hematoxylin & eosin (H&E) staining and luxol fast blue staining, followed by using ImageJ. Luxol fast blue staining was conducted by using the Luxol Fast Blue Stain Kit (Abcam), following the manufacturer’s recommended protocol. Representative histological images were selected for each group of mice.
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