Cresyl violet staining was conducted as described in a previous study (Yoo et al., 2015). Briefly, DRG sections were rinsed in 1% cresyl violet acetate solution (Sigma-Aldrich, St. Louis, MO, USA) containing glacial acetic acid (Sigma-Aldrich). Before and after overnight staining at 25°C, the sections were washed twice in distilled water. Samples were then dehydrated and mounted using Canada Balsam (Kanto, Tokyo, Japan). Digital images of the DRG were captured with a BX51 light microscope (Olympus, Tokyo, Japan) equipped with a digital camera (DP72, Olympus) connected to a computer monitor.
Cresyl violet acetate solution
Manufactured by Merck Group
Sourced in United States
Cresyl violet acetate solution is a laboratory reagent used for staining and visualization purposes. It is a synthetic dye that can be used to stain certain cellular components in biological samples. The solution contains cresyl violet acetate dissolved in a suitable solvent.
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Lab products found in correlation
Epifluorescent microscope, by Zeiss (2 mentions)
Canada balsam, by Kanto Chemical (2 mentions)
Ntb emulsion, by Kodak (2 mentions)
D 19 developer, by Kodak (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
Bx51 light microscope, by Olympus (1 mentions)
Fixer, by Kodak (1 mentions)
Dmrb microscope, by Olympus (1 mentions)
Poly l lysine coated slide, by Thermo Fisher Scientific (1 mentions)
Adhesive caps, by Zeiss (1 mentions)
Palm microbeam system, by Zeiss (1 mentions)
Pen membrane covered slides, by Zeiss (1 mentions)
H e staining kit, by Vector Laboratories (1 mentions)
Histocore autocut, by Leica (1 mentions)
Ss32 500, by Thermo Fisher Scientific (1 mentions)
Cellsens imaging software, by Olympus (1 mentions)
Cm1950, by Leica (1 mentions)
11 protocols using cresyl violet acetate solution
1
Cresyl Violet Staining of Dorsal Root Ganglia
2
Histochemical Analysis of Neuronal Damage
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To elucidate the neuronal death/damage induced by MI, we performed CV and F-J B histofluorescence staining as previously described (Lee et al., 2010). In brief, the sections were stained with cresyl violet acetate solution (1.0% (w/v), Sigma-Aldrich, St. Louis, MO, USA), dehydrated in a graded ethanol series, cleared in xylene, and coverslipped. They were then mounted with Canada balsam (Kanto chemical, Tokyo, Japan). For F-J B histofluorescence, the sections were immersed in a F-J B (0.0004%, Histochem, Jefferson, AR, USA) staining solution. After washing, the sections were investigated using an epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue (450–490 nm) excitation light and a barrier filter. F-J B-positive cells were counted in a 250 × 250 μm2 square, selected approximately at the center of the cingulate cortex and the piriform cortex. Cell counts were obtained by averaging the total cell numbers from each rat per group.
3
Radioactive In Situ Hybridization for Gene Expression
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We followed a previously described protocol published in detail in [71 (link)]. In brief, first, sections were fixed in 3% paraformaldehyde, rinsed in 1× phosphate buffer saline (PBS), dehydrated in an increasing ethanol series, and then air-dried. We generated radioactive 35S-UTP-labeled sense and antisense riboprobes by reverse transcription, and hybridized each slide containing brain sections with 1 × 106 cpm at 60°C (PV; the lower than 65°C temperature is to obtain cross-species hybridization). We exposed the slides to NTB emulsion (Kodak, USA) diluted 1:1 in distilled water for approximately 14 to 30 d at 4°C and processed the slides with D-19 developer (Kodak) and fixer (Kodak). We visualized the bound riboprobe as silver grains, and the cell bodies by counterstaining with Cresyl violet acetate solution (Sigma, USA). Tissue incubated with the sense probe showed no significant signal above background.
4
Quantitative Analysis of Neat1 Expression in Mouse Brain
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Whole mouse brains and spinal cord sections were fixed in 4% PFA overnight and embedded in paraffin wax. Eight µm thick sections were mounted on poly-l -lysine coated slides (ThermoScientific). For RNAscope® ISH analysis, Neat1_1 (440351), with the target region 1416–2381 in mouse Neat1, and Neat1_2 (538761), with the target region 7378–8453, probes (Advanced Cell Diagnostics) were used according to manufacturer’s instructions. For Nissl staining, sections were incubated in 0.5% Cresyl Violet Acetate solution (C5042, Sigma) and differentiated in acidified ethanol. Immunohistochemistry with 3,3′-diaminobenzidine (DAB) as a substrate was performed as described earlier29 (link). Images were taken using Leica DMRB microscope or Olympus BX40 slide scanner (×20 magnification). The images of sagittal sections for the same brain regions (1000 ± 200 µm from the midline) for all animals were obtained. Quantification of neuron numbers was performed using the 3D Object Counter plugin of ImageJ using an equally sized region of interest (ROI) for the cortex zone incorporating all cortex layers.
5
In Situ Localization of PVALB and SLIT1 mRNA
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To localize mRNA expression of the PVALB and SLIT1 genes we used radioactive 35S in situ hybridization following previously described procedures [54 (link),83 (link)]. To make the PVALB riboprobe we used a clone from our zebra finch full-length cDNA collection [38 (link), 46 (link)] (GenBank Accession # DQ215755), and a SLIT1 clone containing 1.8Kb of the coding region that we cloned [39 (link)] (GenBank Accession # KF738084). Sections were fixed in 3% paraformaldehyde, rinsed in 1X phosphate-buffer saline (PBS), dehydrated in an increasing ethanol series, and then air-dried. We generated radioactive 35S-UTP-labeled sense and antisense riboprobes by reverse transcription, and hybridized each slide containing brain sections with 1x106 cpm at 60°C (PVALB, SLIT1; the lower than 65°C temperature is to obtain cross-species hybridization). We exposed the slides to NTB emulsion (Kodak, USA) diluted 1:1 in distilled water for ~14–30 days at 4°C and processed the slides with D-19 developer (Kodak) and fixer (Kodak). We visualized the bound riboprobe as silver grains, and the cell bodies by counterstaining with cresyl-violet acetate solution (Sigma, USA). Tissue incubated with the sense probe showed no significant signal above background.
6
Laser Capture Microdissection of Intestinal Tissues
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Laser capture microdissection (LCM) was performed on 12 μm cryosections mounted on UV-light pretreated PEN-membrane covered slides (Carl Zeiss, Jena, Germany). Tissue sections were shortly air-dried and stored at -80°C for LCM. Prior to LCM, cryosections were thawed and stained with RNase-free 1% (w/v) cresyl violet acetate solution (Sigma) according to Carl Zeiss MicroImaging PALM protocols. LCM was performed with the PALM MicroBeam System (Carl Zeiss MicroImaging, Bernried, Germany) according to the manufacturer’s instructions. Captured tissue segments were collected in AdhesiveCaps (Carl Zeiss) until a total tissue area of on average 2.1x106 μm2 for myenteric and submucosal structures and of 2.8x106 μm2 for mucosal segments was excised. Segments were excised as directly adjacent oval shapes, thereby the enteric myenteric and submucosal regions of the cryosections were collected. Prior to RNA isolation lysis buffer was added and AdhesiveCaps were incubated upside down for 30 min at room temperature. RNA quality from randomized representative samples was assessed in preliminary experiments using the Experion RNA HighSens Assay (Biorad) and yielded good results in both the muscular and mucosal samples (average RIN 7.4).
7
Assessing Ischemia-Induced Neuronal Degeneration
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To find the neuronal death/damage induced by ischemia/reperfusion, we performed CV and F-J B histofluorescence staining as previously described (Lee et al., 2014). In brief, the sections were stained with cresyl violet acetate solution (1.0% (w/v), Sigma-Aldrich, St. Louis, MO, USA), dehydrated in a graded ethanol series, cleared in xylene, and coverslipped. The stained sections were mounted with Canada balsam (Kanto chemical, Tokyo, Japan). For F-J B histofluorescence, the sections were immersed in a F-J B (0.0004%, Histochem, Jefferson, AR, USA) staining solution. After washing, the sections were investigated using an epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue excitation light (450–490 nm) and a barrier filter. With this tool, neurons that undergo degeneration brightly fluoresce in comparison to the background (Schmued and Hopkins, 2000).
8
Brain Tissue Histopathological Staining
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For histopathological staining, the brain tissue samples were embedded in paraffin and cut serially into 4 μm-thick axial sections using a microtome (HistoCore AUTOCUT; Leica, Wetzlar, Germany). Hematoxylin and eosin (H&E) and cresyl violet staining were conducted to identify tissue/neuronal damage post-sonication. Paraffin-embedded sections were deparaffinized and rehydrated prior to staining. Brain tissue slides were stained using an H&E staining kit (Vector Laboratories, Burlingame, CA, USA) or 0.1% cresyl violet acetate solution (Sigma-Aldrich).
9
Cresyl Violet Staining of Cerebellum
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The serial sections of the cerebellum were mounted on Poly-L-lysine-coated adhesive glass slides and allowed to dry over a slide warmer. The sections were transferred to a slide rack and put in a chloroform/ethanol (4:1) solution for 1 h and then placed in cresyl violet acetate solution (Sigma-Aldrich, Germany) for 10 min, with careful monitoring of the colour of the tissue, to avoid overstaining. They were dehydrated by passing through ascending concentrations of ethanol, 50%, 70%, 80%, 90%, 96% and 100% twice for 1 minute each. The sections were then passed through two changes of xylene for 10 minutes each, cover-slipped with DPX Mountant (BDH Chemicals Ltd., England) and allowed to dry.
10
Histochemical staining of tissue sections
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