Cresyl violet

Manufactured by Thermo Fisher Scientific

Sourced in United States

Cresyl violet is a laboratory dye commonly used in histology and neuroscience. It is a purple-colored stain that selectively binds to Nissl bodies, which are accumulations of rough endoplasmic reticulum in the cytoplasm of neurons. This staining technique allows for the visualization and identification of neuronal cell bodies in tissue samples.

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23 protocols using cresyl violet

Cresyl Violet Staining of Tissue

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Every 12^th sections was rinsed in TBS, mounted onto glass
slides, dried, and stained with cresyl violet (Acros Organics, Morris
Plains, New Jersey): tissue was rinsed in decreasing concentrations of
ethanol followed by distilled water (dH₂0) and cresyl violet
(0.1% in Walpole Buffer) for 3 minutes. After a quick rinse in
dH₂0, tissue was processed through increasing concentrations
of ethanol followed by clearing in xylenes (Leasure and Nixon, 2010 (link)).

Hayes D.M., Nickell C.R., Chen K.Y., McClain J.A., Heath M.M., Deeny M.A, & Nixon K. (2018). Activation of neural stem cells from quiescence drives reactive hippocampal neurogenesis after alcohol dependence. Neuropharmacology, 133, 276-288.

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Laser Capture Microdissection of Arcuate Nucleus

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Brains were collected from mice after 2 days or 4 weeks of Cort treatment. Brains were snap frozen and stored at −80 °C before cryosectioning. Consecutive coronal sections (2 day, 30 μm; 4 week, 12 μm) encompassing the whole ARC (−1.46 to −2.7 mm caudal to bregma [21] ) were mounted onto RNAse-free membrane (2 day; MMI) or Superfrost™ Plus slides (4 week; Thermo Scientific). Frozen sections were fixed in 95% ethanol and rehydrated in 75% and 50% ethanol. Slides were stained with cresyl violet to reveal the hypothalamic cytoarchitecture (Ambion), then dehydrated in a graded ethanol series and left to air dry prior to dissection. The entire ARC was dissected from 4 week samples using the PALM Microbeam Laser Capture Microdissection System (Zeiss), and from 2 day samples using an MMI CellCut Laser Microdissection System mounted onto an IX83 inverted microscope (Olympus). RNA was extracted using an RNeasy Micro Plus Kit (Qiagen) according to manufacturer's instructions and analyzed for RNA integrity on an Agilent 2100 Bioanalyzer using an RNA 6000 Pico Kit. All RNA that was subsequently amplified had RNA integrity (RIN) scores >6.5.

Wray J.R., Davies A., Sefton C., Allen T.J., Adamson A., Chapman P., Lam B.Y., Yeo G.S., Coll A.P., Harno E, & White A. (2019). Global transcriptomic analysis of the arcuate nucleus following chronic glucocorticoid treatment. Molecular Metabolism, 26, 5-17.

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Isolation and Microdissection of Murine Pancreatic Tissues

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Pancreata were isolated from aged KPC (4–20 weeks) or WT C57BL/6 mice and fresh-frozen in Tissue-Tek OCT (Sakura Finetek USA) or formalin-fixed and paraffin-embedded (FFPE) by the Johns Hopkins University Oncology Tissue Services. Guide slides were sectioned and stained with H&E by the Johns Hopkins University Oncology Tissue Services. Guide slides containing various grades of PanINs were identified by a pathologist. 10, 10 um frozen sections adjacent to guide sections were mounted on membrane slides (Zeiss) and stained with Cresyl Violet (Ambion). FFPE sections were stained with the Arcturus Paradise Plus Reagent System Staining kit (Thermo Fisher Scientific). Normal, low grade PanIN1, high grade PanIN2/3, and PDA tissue were microdissected using LCM (Leica LMD7000).

Chu N.J., Anders R.A., Fertig E.J., Cao M., Hopkins A.C., Keenan B.P., Popovic A., Armstrong T.D., Jaffee E.M, & Zimmerman J.W. (2020). Inhibition of miR-21 regulates mutant KRAS effector pathways and intercepts pancreatic ductal adenocarcinoma development. Cancer prevention research (Philadelphia, Pa.), 13(7), 569-582.

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Laser Capture Microdissection of Mouse Brain Nuclei

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Fresh frozen brains were collected from Nnat^+/+ and Nnat^+/−p male mice between 13 and 14 weeks of age. Coronal sections of 20-μm thickness were prepared on a cryostat (Bright OTF5000 cryostat) and mounted on RNase-free membrane-coated glass slides (Superfrost Plus, Thermo Fisher Scientific). Brain sections were fixed in 95% ethanol (30 s) and rehydrated in an ethanol series (75% ethanol, 50% ethanol) prior to staining with cresyl violet (Ambion). Sections were then dehydrated in another ethanol series of 50%, 75%, 95%, and 100% ethanol and left to air dry prior to laser capture. PVN and ARC, were then dissected using a PALM Microbeam Laser Capture Microdissection System (Zeiss) on a × 5 objective as previously described^{48 (link)}. Briefly, tissues were captured on AdhesiveCap Clear PCR tubes (Zeiss) and stored in 80 μl of QIAzol (QIAGEN) on dry ice prior to RNA extraction. Laser capture and subsequent cDNA library preparation were performed. RNA from laser-captured nuclei was extracted using the miRNeasy Micro RNA Extraction Kit (QIAGEN). Quality and quantity of the samples were then checked on an Agilent 2100 Bioanalyzer using Agilent RNA 600 pico and/or nano chips, with a typical sample RNA concentration in the range of 1–5 ng/μl.

Cimino I., Rimmington D., Tung Y.C., Lawler K., Larraufie P., Kay R.G., Virtue S., Lam B.Y., Fagnocchi L., Ma M.K., Saudek V., Zvetkova I., Vidal-Puig A., Yeo G.S., Farooqi I.S., Pospisilik J.A., Gribble F.M., Reimann F., O’Rahilly S, & Coll A.P. (2021). Murine neuronatin deficiency is associated with a hypervariable food intake and bimodal obesity. Scientific Reports, 11, 17571.

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Cryosectioning and Staining of Mouse Bone

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Mice were sacrificed by cervical dislocation. Mandible and tibia were immediately excised, and the specimen were embedded in OCT compound (Sakura Fineteck Japan, Tokyo, Japan) and rapidly frozen in isopentane cooled by liquid nitrogen. Undecalcified nonfixed serial frozen sections (5 μm-thick) were prepared using a super-hard tungsten steel knife (Meiwa Shoji Ltd., Tokyo, Japan) in − 25 °C cryostat (Leica Microsystems, Wetzlar, Germany) (Nakamura et al., 2007 (link)). The sections were collected individually on a 1.35 μm thick polyethylene naphthalene membrane with an adhesive (SECTION-LAB Ltd., Hiroshima, Japan) (Kawamoto and Shimizu, 1986 (link)) and stuck onto slides. Then the sections were slightly fixed with nuclease-free 95% and 75% ethanol for 30–40 s. each, and 50% for 25–30 s. Next, they were stained with Cresyl Violet which provided as LCM Staining Kit (Ambion, Austin, TX) for 40 s., and they were washed through nuclease-free 50% and 75% ethanol for 25–30 s. each, and 95% and 100% for 30–40 s. each.

Miyamoto Y., Kanzaki H., Wada S., Tsuruoka S., Itohiya K., Kumagai K., Hamada Y, & Nakamura Y. (2017). Asporin stably expressed in the surface layer of mandibular condylar cartilage and augmented in the deeper layer with age. Bone Reports, 7, 41-50.

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COPD Epithelial Microdissection and RNA Extraction

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Optimal Cutting Temperature (OCT)-embedded proximal bronchial tissue from non-smoker controls (n = 5) and very severe COPD patients (n = 6) was selected. The day before microdissection, 20 µm thick sections were obtained, in RNase-free conditions on a microtome-cryostat (cryochamber temperature, −20 °C). Sections were disposed on polyethylene naphtalate (PEN) Membrane Frame Slides (Thermo Fisher Scientific) and stored at −80 °C. Less than an hour before microdissection, sections were dehydrated in graded ethanol solutions, stained with cresyl violet (Ambion) and fixed with xylene. Epithelial microdissection was performed using the Leica LMD 6000. RNA was extracted from microdissected samples as previously described [43] (link). Detailed information on sample preparation, staining and microdissection is provided elsewhere [46] (link).

Carlier F.M., Dupasquier S., Ambroise J., Detry B., Lecocq M., Biétry–Claudet C., Boukala Y., Gala J.L., Bouzin C., Verleden S.E., Hoton D., Gohy S., Bearzatto B, & Pilette C. (2020). Canonical WNT pathway is activated in the airway epithelium in chronic obstructive pulmonary disease. EBioMedicine, 61, 103034.

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Fibrotic Liver Tissue Isolation and Analysis

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Cryosections from liver tissues at 1 month after BDL and non-treated normal livers (n = 3/3 rats) were stained by Cresyl Violet (Ambion) to visualize the fibrotic septa and surrounding parenchymal tissue regions, which was followed by laser capture microdissection (LCM) using the LMD6500 Laser Microdissection System (Leica Microsystems). By collecting laser-captured liver tissue samples from 5–10 fibrotic septa regions and equally sized regions from surrounding parenchyma of each rat, we isolated 50–150 ng RNA/sample using the PicoPure RNA isolation kit (Life Technology). LCM-derived RNA had a very high integrity without DNA contamination (RIN numbers were between 8.0 and 9.3), as determined using the 2100 Bioanalyzer system (Agilent Technologies). RNA was amplified using the Ovation PicoSL WTA System V2 (NuGEN Technologies). After one round of amplification, at least 7 μg complementary DNA/sample was obtained, subsequently pooled for each group and used for RT-PCR, qRT-PCR analysis for selected genes or PCR array analysis.

Yovchev M.I., Locker J, & Oertel M. (2016). Biliary fibrosis drives liver repopulation and phenotype transition of transplanted hepatocytes. Journal of hepatology, 64(6), 1348-1357.

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Laser Capture Microdissection and RT-PCR Analysis

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Tissue sections (10 μm) were placed on RNase-free slides, dehydrated in graded ethanols, cleared in xylene, stained with cresyl violet (Ambion, Life Technologies, Grand Island, NY) and dehydrated in graded ethanols and two washes in xylene. Laser capture microdissection was performed with an Arcturus(XT) laser capture microdissection instrument (Arcturus Engineering, Inc., Mountain View, CA), using CapSure HS caps (Arcturus MDS Analytical Technologies, Sunnyvale, CA). The cap film, with microdissected tissue, was immediately placed in TRIzol reagent (Invitrogen). RNA was isolated and cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). The cDNA was used as template for RT-PCR using JumpStart Taq polymerase (Sigma-Aldrich, St. Louis, MO): (hot start, 94°C, 3 min followed by 40 cycles of 94°C, 30 sec; 56°C, 45 sec; 72°C, 1 min; and a final extension, 72°C, 5 min). Rabbit CXCL12 and HGPRT (control for cDNA quality) PCR primers are listed above. One-tenth volume of each PCR product was electrophoresed on a 5% PAGE gel, stained with ethidium bromide and scanned on a Typhoon imager (GE Healthcare Biosciences, Pittsburgh, PA).

Zhai S.K., Volgina V.V., Sethupathi P., Knight K.L, & Lanning D.K. (2014). Chemokine-mediated B cell trafficking during early rabbit GALT development. Journal of immunology (Baltimore, Md. : 1950), 193(12), 5951-5959.

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Rodent Brain Extraction and Sectioning

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In experiments 1–4, rats were overdosed with ketamine hydrochloride and xylazine (100 and 5 mg/kg, i.v., or 300 and 15 mg/kg, i.p., respectively, depending on catheter patency). The brains were extracted, rapidly frozen in methyl butane, and stored at −80 °C. Brains were sectioned in the coronal plane at 50 μm on a cryostat (Leica Biosystems, Buffalo Grove, Illinois, USA). Sections were mounted on glass slides and stained with cresyl violet (Fisher Scientific, Waltham, Massachusetts, USA). The most ventral portion of each cannula tract was identified under a light microscope. The data of rats with cannula placements outside of the BLA were excluded from all statistical analyses.

Ritchie J.L., Walters J.L., Galliou J.M., Christian R.J., Qi S., Savenkova M.I., Ibarra C.K., Grogan S.R, & Fuchs R.A. (2021). Basolateral amygdala corticotropin-releasing factor receptor type 1 regulates context-cocaine memory strength during reconsolidation in a sex-dependent manner. Neuropharmacology, 200, 108819.

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Preserving Brain Tissue for Histological Analysis

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After the surgery and the data recording, the rats were euthanized with a 1 ml injection of ketamine and decapitated for the removal of the brain. The brain was stored at a low temperature in a 10% formalin solution (Fisher Scientific) in a glass vial. The formalin solution in the glass vial was exchanged for a 20% sucrose solution (Fisher Scientific), prior to sectioning. After removing the brain from the sucrose solution and freezing it with dry ice, a freezing microtome (Microm HM 450, Thermo Scientific) was used for slicing sections 50 μm thick. The brain slices were separated according to the rat brain atlas in a petri dish containing PBS, and then mounted on Superfrost plus microscope slides using gelatin buffer [Gelatin type A (Acros), chromium (III) potassium sulfate (Fisher Science Education), Sodium Azide (Fisher Scientific)]. The slices were left to dry before being stained in Cresyl Violet (Fisher Scientific) staining. After drying from the staining, the slides were cover-slipped with Permaslip (Alban Scientific Inc.) for preservation and a light microscope was used for the analyses of the electrode placements.

Sibilska S., Mofleh R, & Kocsis B. (2023). Development of network oscillations through adolescence in male and female rats. Frontiers in Cellular Neuroscience, 17, 1135154.

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