Luxol fast blue (lfb)

Manufactured by Thermo Fisher Scientific

Sourced in United States

Luxol fast blue is a histological stain used to identify myelin in tissue samples. It selectively stains the lipid-rich myelin sheaths surrounding nerve fibers, allowing for the visualization and analysis of the structure and integrity of the nervous system.

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Lab products found in correlation

Celltracker cm dii, by Thermo Fisher Scientific (2 mentions) Affinipure goat anti rabbit, by Jackson ImmunoResearch (2 mentions) Dl 1174, by Vector Laboratories (2 mentions) Anti albumin, by ABclonal (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Lithium carbonate, by Thermo Fisher Scientific (2 mentions) Hhs128, by Merck Group (2 mentions) Bz x810 microscope, by Keyence (2 mentions) Hematoxylin eosin, by Thermo Fisher Scientific (1 mentions) Fisher brand superfrost plus glass slides, by Thermo Fisher Scientific (1 mentions) Luxol fast blue (lfb), by Merck Group (1 mentions) Bright field, by Olympus (1 mentions) Glacial acetic acid, by Thermo Fisher Scientific (1 mentions) Dpx mounting medium, by Electron Microscopy Sciences (1 mentions) Bx51 microscope, by Olympus (1 mentions) Hematoxylin and eosin, by Thermo Fisher Scientific (1 mentions) Slidepath digital image hub, by Leica camera (1 mentions) Scn400 slide scanner, by Leica camera (1 mentions) Alexa fluor 594, by Proteintech (1 mentions) Anti lc3ii antibody, by Cell Signaling Technology (1 mentions)

13 protocols using luxol fast blue (lfb)

Histological Processing of Neural Tissues

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Following fixation in 10% buffered formalin, coronal sections of brain tissue, axial sections of spinal cord, and longitudinally-oriented optic nerves were processed and embedded in paraffin blocks. 4 μm sections were cut, mounted on Fisher Brand Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA), and stained with hematoxylin & eosin (Fisher Scientific).
For the Luxol Fast Blue stained-sections, 6 μm thick sections of paraffin-embedded tissue were cut on a rotary microtome and mounted on Fisher Brand Superfrost Plus glass slides (Fisher Scientific). The sections were deparaffinized and hydrated. Following heating of the sections in 0.1% Luxol Fast Blue (Sigma-Aldrich, St. Louis, MO) at 60°C for at least one hour, excess stain was rinsed off. They were then differentiated in 70% alcohol for 25 seconds and rinsed in distilled water. Next, the sections were evaluated under the microscopy and depending on the adequacy of differentiation they were subjected to an additional round of lithium carbonate, and an alcohol rinse.

Arellano B., Hussain R., Miller-Little W.A., Herndon E., Lambracht-Washington D., Eagar T.N., Lewis R., Healey D., Vernino S., Greenberg B.M, & Stüve O. (2016). A Single Amino Acid Substitution Prevents Recognition of a Dominant Human Aquaporin-4 Determinant in the Context of HLA-DRB1*03:01 by a Murine TCR. PLoS ONE, 11(4), e0152720.

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Cerebellar and Cortical Pathology in SCA3

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All available post-mortem human cerebellum and cortex paraffin tissue from patients with SCA3 and control subjects (cause of death was not CNS related) were acquired from the Michigan Brain Bank (Table 1). Sectioned tissues (5 μm) were deparaffinized and stained with Luxol Fast Blue MBSN (Matheson Coleman & Bell, Norwood, OH, United States). Briefly, after dewaxing sections in xylene, slides were rinsed in 100% then 95% ethanol. Sections were stained for approximately 16 h with 0.1% Luxol Fast Blue in 95% ethanol and 1:200 glacial acetic acid (Fisher Scientific, Hampton, NH, United States). After staining, sections were rinsed in 95% ethanol and distilled water. Finally, slides were sequentially rinsed in 70% ethanol, distilled water, then 90 and 100% ethanol before being fixed in xylene and cover-slipped (Corning, NY) with DPX mounting medium (Electron Microscopy Sciences, Hatfield, PA, United States). Images were taken on a bright-field BX51 microscope (Olympus, Center Valley, PA, United States) at 10× and 20× magnification.

Schuster K.H., DiFranco D.M., Putka A.F., Mato J.P., Jarrah S.I., Stec N.R., Sundararajan V.O, & McLoughlin H.S. (2023). Disease-associated oligodendrocyte signatures are spatiotemporally dysregulated in spinocerebellar ataxia type 3. Frontiers in Neuroscience, 17, 1118429.

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Histological Analysis of Spinal Cords

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Spinal cords of MOG-immunized mice were imbedded in paraffin and serial sections were cut at 5 μm. Slides were stained with hematoxylin and eosin (Thermo Scientific) and 0.1% Luxol fast blue (Acros Organics) in 95% ethanol using standard methods. Images were acquired using a SCN400 slide scanner (Leica) and viewed by Slidepath Digital Image Hub (Leica).

Kaufmann U., Shaw P.J., Kozhaya L., Subramanian R., Gaida K., Unutmaz D., McBride H.J, & Feske S. (2015). Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function. Journal of immunology (Baltimore, Md. : 1950), 196(2), 573-585.

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Multimodal Assessment of Blood-Brain Barrier

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All chemicals and antibodies used in the studies were bought from reputed commercial vendors. Anti-albumin (A0353, ABClonal) and FITC-conjugated AffiniPure Goat Anti-Rabbit (111–095-003, Jackson Laboratories) were used to determine the leakage of BBB. Luxol Fast Blue (#212170250, Acros Organics) used to assess the integrity of white matters, Prussian Blue Stain kit (#3160, Eng Scientific) was used to detect micro bleeding in the brain. HEK293 cells were acquired from ATCC, and mouse endothelial progenitor cells (EPC) were collected from the bone marrow of mice using a magnetic sorter as per our previous methods [28 (link)]. CellTracker^™ CM-DiI (C7000, Thermo Fisher) lipophilic fluorescent dye was purchased for exosome labeling. Fluorescent (FITC) tagged tomato lectin was used to outline the blood vessels (DL-1174, Vector Labs).

Federici L., Caprari C., Mattei B., Savino C., Di Matteo A., De Lorenzo G., Cervone F, & Tsernoglou D. (2001). Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein). Proceedings of the National Academy of Sciences of the United States of America, 98(23), 13425-13430.

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Multimodal Assessment of Brain Barrier Integrity

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All chemicals and antibodies used in the studies were bought from reputed commercial vendors. Anti-albumin (A0353, ABClonal) and FITC-conjugated AffiniPure Goat Anti-Rabbit (111-095-003, Jackson Laboratories) were used to determine the leakage of BBB. Luxol Fast Blue (#212170250, Acros Organics) used to assess the integrity of white matters, Prussian Blue Stain kit (#3160, Eng Scientific) was used to detect micro bleeding in the brain. HEK293 cells were acquired from ATCC, and mouse endothelial progenitor cells (EPC) were collected from the bone marrow of mice using a magnetic sorter as per our previous methods [28] . CellTracker ™ CM-DiI (C7000, Thermo Fisher) lipophilic fluorescent dye was purchased for exosome labeling. Fluorescent (FITC) tagged tomato lectin was used to outline the blood vessels (DL-1174, Vector Labs).

Alptekin A., Khan M.B., Ara R., Rashid M.H., Kong F., Parvin M., Frank J.A., Chopra R., Dhandapani K, & Arbab A.S. (2021). Pulsed Focal Ultrasound as a Non-Invasive Method to Deliver Exosomes in the Brain/Stroke. Journal of biomedical nanotechnology, 17(6).

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Histological Assessment of EAE Pathology

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Mice were sacrificed at day 21 after immunization and the lumbar segment of spinal cords from EAE mice pretreated with PTL or DMSO were removed and fixed in 4% paraformaldehyde. Fixed spinal cords were embedded with paraffin and cut into 10-μm sections. Hematoxylin-eosin (H&E) and Luxol fast blue (Alfa Aesar, Ward Hill, USA) were used to examine the inflammatory infiltration and demyelination, respectively. The percentage of inflammatory infiltration and demyelination areas were measured by software ImageJ.

Zhang Z., Zhang K., Zhang M., Zhang X, & Zhang R. (2022). Parthenolide Suppresses T Helper 17 and Alleviates Experimental Autoimmune Encephalomyelitis. Frontiers in Immunology, 13, 856694.

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Histological Assessment of Spinal Cord Inflammation

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The spinal cords from mice transcardially perfused with 4% paraformaldehyde were dissected and postfixed overnight. Paraffin-embedded 5–10 μm spinal cord sections were stained with hematoxylin-eosin (H&E) for routine histological analysis of inflammatory infiltration and with Luxol fast blue (Alfa Aesar, Ward Hill, USA) for evaluation of demyelination. For confocal imaging of microglia and autophagosome colocalization, the mouse anti-IbaI (Abcam) and anti-LC3II antibody (Cell Signaling Technology) were used for immunostaining. Donkey anti-rabbit and donkey anti-goat conjugated to Alexa Fluor 488 or Alexa Fluor 594 were used as secondary antibodies (all from Proteintech). The Olympus FluoView FV1000 Microscope equipped with a ×100 objective was used for cell imaging. For p65 immunofluorescence, cells were cultured on glass coverslips, mouse anti-p65 (1:400) was the primary antibody and Alexa Fluor 488-labeled goat anti-rabbit antibody (1:1000) was the secondary antibody. Coverslips were mounted and stored at 4 °C for further investigation.

Xue Z., Zhang Z., Liu H., Li W., Guo X., Zhang Z., Liu Y., Jia L., Li Y., Ren Y., Yang H., Zhang L., Zhang Q., Da Y., Hao J., Yao Z, & Zhang R. (2018). lincRNA-Cox2 regulates NLRP3 inflammasome and autophagy mediated neuroinflammation. Cell Death and Differentiation, 26(1), 130-145.

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Characterization of Neuroinflammation and Autophagy in mir223 Knockout Mice

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The lumbar spinal cords from the female C57BL/6 mice and mir223^−/- mice (n = 6) were perfused transcardially with 4% (weight:volume) paraformaldehyde and were then dissected and post-fixed for 48 h. The spinal cord paraffin sections (7 μm) were stained with H&E to detect inflammatory cell infiltration and with luxol fast blue (Alfa Aesar,1328–51-4) to assess demyelination.
The brains from the wild-type C57BL/6 mice and mir223^−/- mice (n = 6) were perfused transcardially with 4% (weight:volume) paraformaldehyde and then were dissected and post-fixed for 48 h. The brain paraffin sections (7 μm) were stained with DAPI (Thermo Fisher Scientific, 00–4959-52), LC3A/B (Cell Signaling Technology, 12,741), BCL2 (Abcam, ab32124), BECN1 (Cell Signaling Technology, 3495), AIF1/IBA1 (allograft inflammatory factor 1) (Abcam, ab107159) and Alexa Fluor 488 (Proteintech, SA00006-2) and Alexa Fluor 546 (Thermo Fisher Scientific, A11056) secondary antibody conjugates (green and red, respectively).

Li Y., Zhou D., Ren Y., Zhang Z., Guo X., Ma M., Xue Z., Lv J., Liu H., Xi Q., Jia L., Zhang L., Liu Y., Zhang Q., Yan J., Da Y., Gao F., Yue J., Yao Z, & Zhang R. (2018). Mir223 restrains autophagy and promotes CNS inflammation by targeting ATG16L1. Autophagy, 15(3), 478-492.

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Spinal Cord Injury White Matter Preservation Assay

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Several sections (3–5) that cover an area of 5 mm within the lesion epicenter, from each animal (n = 3), were stained with cresyl violet (Sigma-Aldrich) to visualize nuclei and with Luxol Fast Blue (Luxol Fast Blue, Alfa Aesar, Heysham, Lancashire, UK) for myelin as previously described (Figueroa et al., 2006; Santiago et al., 2009). The selection of sections in this range of 5 mm within the lesion epicenter provides an overview of the area and any effect of PP2 in white matter spared tissue. Once the counter-stain assay was done, the sections were visualized using a Digital Microscope (Fisher Scientific, Pittsburgh, PA, USA) and photomicrographs were taken using the Motic version 1.2 professional software. The stained spinal cord sections were morphometrically analyzed with ImageJ 1.43u® software (Wayne Rashband) to determine the extent of white matter spared tissue. To calculate the area of remnant tissue, the outer border of the spinal cord and the inner border of the lesion cavity, including the remainder of the grey matter, were delineated and the redundant area was substracted from the total (outer border) area.

Rosas O.R., Torrado A.I., Santiago J.M., Rodriguez A.E., Salgado I.K, & Miranda J.D. (2014). Long-term treatment with PP2 after spinal cord injury resulted in functional locomotor recovery and increased spared tissue. Neural Regeneration Research, 9(24), 2164-2173.

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Histological Analysis of Spinal Cord Pathology

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On 30 DPI, five animals per group were euthanized by exposure to CO₂ and perfused with sterile PBS. Spinal cords were collected, fixed in 10% formalin (Thermo Fisher Scientific) and embedded in paraffin. Lymph nodes and spleens were collected, weighed, fixed in formalin, and paraffin embedded. Tissue sections were cut at 6 μm thickness on a microtome, and where indicated, sections were stained for H&E (Thermo Fisher Scientific) to reveal inflammatory infiltrates or Luxol fast blue for myelin levels (IHC World, Ellicott City, MD, www.ihcworld.com). Histological images were acquired with Scan-Scope (Aperio, Vista, CA, www.leicabiosystems.com), and histological analyses were conducted with ImageScope (Aperio). An index of cellular content was determined by the percentage of positive pixels divided by the total number of pixels in the section. The demyelination score was calculated by the area of intact myelin against the total number of pixels in the section. The number of lesions in each spinal cord section was counted and the surface area occupied by the lesions was acquired.

Strong A.L., Bowles A.C., Wise R.M., Morand J.P., Dutreil M.F., Gimble J.M, & Bunnell B.A. (2016). Human Adipose Stromal/Stem Cells from Obese Donors Show Reduced Efficacy in Halting Disease Progression in the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis. Stem cells (Dayton, Ohio), 34(3), 614-626.

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