Luxol Fast Blue/Cresyl violet (CV) double staining was used to quantify the lesion volume.65 (link) Briefly, the brain sections in series (16-μm-thick) were mounted on slides, immersed in 70% ethanol for 24 h and 0.1% Luxol Fast Blue staining solution (S3382, Sigma-Aldrich, USA) at 56°C overnight. The sections were rinsed with PBS and decolorized in 0.05% lithium carbonate solution (62470, Sigma-Aldrich). Then, the sections were counterstained with 0.2% CV (C5042, Sigma-Aldrich) and dehydrated by a serial increased concentration of ethanol. The slides were immersed in xylene and mounting medium and then observed under a light microscope (Eclipse Ci, Nikon). ImageJ software was used to quantify the lesion volume was calculated. We photographed all brain sections of each mouse at an interval of 96 μm for quantification. The total volume of hematoma was calculated and used for statistical analysis. When comes to CV staining, at least twelve locations per mouse in the perihematomal region [4 fields (upper, lower, left, and right) per section × 3 sections (bregma 1.70 mm, 0.74 mm, and −0.46 mm)] for quantification. The mean value of the twelve fields was calculated and used for statistical analysis.
Lithium carbonate solution
Manufactured by Merck Group
Sourced in Germany
Lithium carbonate solution is a laboratory reagent used in various analytical and experimental procedures. It is an aqueous solution containing lithium carbonate, a salt of the element lithium and carbonic acid. The core function of this product is to provide a source of lithium ions in a liquid form, which can be utilized in different chemical analyses and research applications.
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Lab products found in correlation
Luxol fast blue solution, by Merck Group (2 mentions)
Luxol fast blue (lfb), by Merck Group (2 mentions)
C5042, by Merck Group (1 mentions)
Eclipse ci, by Nikon (1 mentions)
Eosin y solution, by Merck Group (1 mentions)
Nanozoomer digital slide scanner, by Hamamatsu Photonics (1 mentions)
Superfrost plus, by Avantor (1 mentions)
Entallan, by Merck Group (1 mentions)
Entellan, by Merck Group (1 mentions)
Dp12 digital camera, by Olympus (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
9 protocols using lithium carbonate solution
1
Quantifying Brain Lesion Volume with Luxol Fast Blue/Cresyl Violet
2
Luxol Fast Blue Myelin Staining
3
Luxol Fast Blue Staining of Rat Brains
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The protocol of Luxol fast blue staining was in accordance with a previous study (Liu et al., 2017). After their behavior test, two rats of each group were deeply anesthetized and brains were immediately removed for processing. The removed brain tissue was paraffin-embedded and cut into 4-μm-thick coronal sections. After de-paraffinization with xylene, the sections in slides needed to be rehydrated with ethanol gradient from 95% to 70%. Afterwards, 1% Luxol fast blue dye (Sigma-Aldrich) was added and the slides incubated overnight in a 60°C Thermostat box. The sections were rinsed with 95% ethanol and double distilled water (ddH2O) followed by incubation with 0.05% lithium carbonate solution (Sigma-Aldrich) for 5 seconds. After two washes with 70% ethanol and ddH2O, the sections were dehydrated with 95% ethanol to 100% ethanol, and cleared with xylene. Images were captured by bright-field and fluorescence microscope.
4
Klüver-Barrera Staining Protocol
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After perfusion with 4% paraformaldehyde, the brain and spinal cord were extracted and embedded in paraffin using an automated Microm HMP110 paraffin tissue processor. Paraffin material was cut into 5 m thick coronal (brain) or transverse (spinal cord) sections. Prior to staining, sections were deparaffinised in xylene and rehydrated in ethanol of decreasing percentage (from 100% to 70%) followed by rinsing with distilled water. Histochemical staining was performed according to Klüver and Barrera (1953) . In short, sections were stained overnight with Luxol Fast Blue solution (Sigma-Aldrich) at 60 • and rinsed in 95% ethanol and distilled water. To wash away non-specific labelling, sections were rinsed with a 0.05% lithium carbonate solution (Sigma-Aldrich) and washed with 70% ethanol. Sections were then stained with a 0.5% Eosin-Y solution (Sigma-Aldrich), washed with distilled water and stained with a 0.25% Chresyl Echt Violet solution (Chresyl violet acetate, Sigma-Aldrich). After these stainings, slides are rinsed with distilled water, dehydrated using an ethanol series with increasing percentage (80-100%), followed by two washes with xylene.
5
Luxol Fast Blue Staining Protocol
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Paraffin sections seven mm in thickness were collected on glass slides (Superfrost Plus, VWR international, Leuven, Belgium) and dried at 37 C. Sections were deparaffinized and hydrated to 95% ethanol and incubated in luxol fast blue solution (0.1% w/v luxol fast blue, Sigma-Aldrich, in 95% ethanol) overnight at 55 C. The sections were then quickly rinsed in 95% ethanol and incubated in lithium carbonate solution (0.05% w/v, Sigma-Aldrich, in distilled H 2 O) for 30 s. Next, the samples were rinsed in 70% ethanol and subsequently in ddH2O. The sections were dehydrated to 100% ethanol and mounted with Entallan (Merck). Whole slides were digitalized using a NanoZoomer digital slide scanner (Hamamatsu), and the slides were analyzed using Hamamatsu software NDPview2.
6
Luxol Fast Blue Staining and Remyelination Scoring
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To stain for myelin content, tissue sections from mice were treated with Luxol fast blue (LFB, Sigma, USA). Sections were stained overnight in LFP at 56°C and washed in 95% ethanol and distilled water to remove excess blue stain. The color was then differentiated (until white matter was easily distinguishable from gray matter) in lithium carbonate solution (Merck, Germany) for 15 sec, followed by distilled water and three washes of 80% alcohol. Slides were passed through fresh xylene (Merck, Germany) twice, mounted with Entellan (Merck, Germany) and cover slipped.3 (link) In order to evaluate remyelination in LFB stained sections of cuprizone demyelination of the corpus callosum in the mice that received progesterone, three blinded readers scored LFB stained sections between zero and three. A score of three is equivalent to totally myelinated corpus callosum whereas zero is equivalent to totally demyelinated corpus callosum. A score of one or two corresponds to one-third or two-third fiber myelination of the corpus callosum, respectively.1 (link)
7
Luxol Fast Blue Quantification of Corpus Callosum Demyelination
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For histological assessment, brains were fixed in 10% paraformaldehyde solution. The degree of demyelination in the corpus callosum was assessed by Luxol Fast Blue (LFB) staining (Sigma, St. Louis, MO, USA). Briefly, paraffin-embedded samples were put on slides, deparaffinized, rehydrated by reducing concentrations of ethanol and placed in the LFB solution (0.01%) at 60 °C. To distinguish between white and gray matter, tissues were differentiated using a lithium carbonate solution (0.05%, Merck, Darmstadt, Germany). The stained tissues were observed and captured using a light microscope (Olympus BX51 microscope with an Olympus DP12 digital camera). The clear-white areas of demyelination relative to the blue normal tissue were measured and quantified using a total of 10 random sections per mouse.
8
Staining Myelin Sheaths for Remyelination Analysis
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To demonstrate shadow lesions or partially remyelinated lesions and their remyelinating properties, staining of the lipoproteins of myelin sheaths was performed with LFB (Gurr #16765, Electron Microscopy Sciences, Hatfield, PA, USA). Sections were first deparaffinized by softening the paraffin on a heating plate (58 °C for 30–60 min) and then in a series of 100% xylene (3 × 10 min), 2 × 5 min 100% ethanol and 5 min in 96% ethanol. Subsequently, tissue sections were incubated in a 0.1% LFB solution in a stove at 58 °C overnight. All sections were washed and differentiated one by one. Washing steps were performed consecutively in 96% alcohol (2–3 s) and milli-Q (MQ) water (3 s). Immediately thereafter, differentiation steps were performed in 0.05% Lithium carbonate solution (Merck Millipore; 554-13-2; 5 s) and 70% ethanol (5–7 s) until decoloring of the cortical GM, whilst WM remained blue. The latter step was performed carefully and was checked microscopically to ensure homogeneity of the LFB staining. The differentiation steps were repeated if needed. The sections were then rinsed in MQ water. Finally, the samples were dehydrated in a series of ethanol 96% (3–5 min), 100% (2 × 5 min) and xylene (3 × 5 min). The sections were mounted with entellan and a coverslip.
9
Luxol Fast Blue Staining of Brain Tissue
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Deparaffinized sections (see above) were incubated in a 0.1% Luxol Fast Blue (LFB; Gurr, UK) solution in a stove (58°C) overnight. Washing steps were performed consecutively in 96% alcohol (2–3 seconds) and milli‐Q (MQ) water (3 seconds). Immediately thereafter, differentiation steps were performed in 0.05% Lithium carbonate solution (5 seconds; Merck Millipore, Germany) and 70% ethanol (5–7 seconds) until the grey matter was colorless and white matter remained blue. The sections were then rinsed in MQ water and dehydrated in a series of ethanol 96% (3–5 minutes), 100% (2 × 5 minutes), and xylene (3 × 5 minutes) and mounted with Entellan and a coverslip.
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