Fixed brain tissues were put into a glass bottle, and the bottleneck was sealed with gauze. Then the rubber tube was inserted to connect running water and the bottle was washed for 24 h. Then the tissues were dehydrated, transparentized, fixed, embedded with hard wax and beeswax (Sigma Company, American) and cut into slices. The protein was mixed with neutral gum (Sigma Company, USA) and applied to two-thirds of the slices. Then the slices were placed in a water bath (CRYSTAL Company, USA) for surface, dried and put into xylene (Sigma Company, American) for dewaxing. Ethanol was used to elute paraffin and xylene. The slices were stained with hematoxylin (Shanghai Rongbai Biotechnology Co., Ltd., China), separated by hydrochloric acid alcohol, and rinsed with running water overnight. The tissues were sliced and soaked in a solution of 0.5%-1% eosin (Shanghai Rongbai Biotechnology Co., Ltd. China), and the slices were taken out and put into 95% ethanol to continue color separation and dehydration. They were dehydrated twice with anhydrous alcohol, and xylene was used for transparency. Finally, they were sealed with a neutral gum.
Neutral gum
Manufactured by Merck Group
Sourced in United States
Neutral gum is a laboratory equipment used for the stabilization and suspension of particles in solution. It is a versatile material that can be used in a variety of scientific applications to maintain the stability and homogeneity of dispersed systems.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti, by Nikon (3 mentions)
Matrigel, by Corning (3 mentions)
Rpmi 1640 medium, by Solarbio (3 mentions)
Xylene, by Merck Group (1 mentions)
Nissl staining solution, by Beyotime (1 mentions)
Ba210t, by Motic (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
Ckx41, by Olympus (1 mentions)
Silver nitrate solution, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
3 3 diaminobenzidine (dab), by Zhongshan Jinqiao Biotechnology (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Tnf α, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Neuronal nuclei (neun), by Novus Biologicals (1 mentions)
10 protocols using neutral gum
1
Histological Tissue Preparation and Staining
2
Nissl Staining of Rat Brain
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The coronal part of the rat brain was loaded onto slides, air‐dried, and then soaked in xylene for 10 min. After gradient alcohol dehydration, the sections were washed with distilled water and stained in 0.5% Nissl staining solution (Beyotime, Shanghai, China) for 10 min at room temperature. The sections were then immersed in 95% ethanol (pH 4.1) for 5 s, followed by fresh 95% ethanol dehydration for 2 min, xylene clearance for 5 min, and neutral gum (Sigma–Aldrich) sealing. Nissl‐positive neurons showed mottled blue‐purple staining with clear nuclei and blue cytoplasm when viewed under a microscope (Olympus).
3
Histological Analysis of Tumor Tissues in Mice
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The tumor tissues of mice were made into paraffin sections. The sections were baked in the microwave oven at 60°C for 2 h. The sections were then deparaffinized in xylene and placed in 100%, 100%, 95%, 85%, and 75% ethanol for 5 min at each stage. The sections were soaked in distilled water and stained with hematoxylin for 5 min and eosin solution for 3 min. Then, the sections were dehydrated in gradient alcohol and soaked in xylene two times, each 10 min. Finally, they were sealed with neutral gum (Sigma), and photos were taken with an ordinary light microscope (BA210T, Motic).
4
In Vitro Cell Invasion and Migration Assay
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Matrigel (Corning) was diluted with Roswell Park Memorial Institute (RPMI)-1640 medium (Solarbio, Beijing, China) and added into a chamber dropwise. Cells suspended in RPMI-1640 medium (1 × 104
cells/mL) were seeded into 6-well plates, whereas 500 µL of RPMI-1640 medium supplemented with 10% fetal bovine serum was placed in a basolateral chamber. After incubation for 24 h at 37°C and 5% CO2, cells in the basolateral chamber were washed with PBS, fixed in 4% paraformaldehyde for 30 min at room temperature, and stained with 0.5% crystal violet staining solution for 30 min. After fixation with neutral gum (Sigma-Aldrich, St. Louis, MO, USA), 5 visual fields were randomly selected under an inverted microscope (Eclipse Ti, Nikon, Tokyo, Japan) to photograph and count invaded cells. Except for the omission of Matrigel, the cell migration assay was performed using the same steps as in the invasion assay.
cells/mL) were seeded into 6-well plates, whereas 500 µL of RPMI-1640 medium supplemented with 10% fetal bovine serum was placed in a basolateral chamber. After incubation for 24 h at 37°C and 5% CO2, cells in the basolateral chamber were washed with PBS, fixed in 4% paraformaldehyde for 30 min at room temperature, and stained with 0.5% crystal violet staining solution for 30 min. After fixation with neutral gum (Sigma-Aldrich, St. Louis, MO, USA), 5 visual fields were randomly selected under an inverted microscope (Eclipse Ti, Nikon, Tokyo, Japan) to photograph and count invaded cells. Except for the omission of Matrigel, the cell migration assay was performed using the same steps as in the invasion assay.
5
Silver Staining Assay for Cell Nuclei
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Cells from each group were seeded in 24-well culture plates containing cover slips. The culture plates were placed in an incubator at 37°C, with 5% CO2. When cultivated cells grew to a mono-layer, the cover slips were removed and cells were fixed in 95% ethanol. The samples were immersed in deionized water for hydration; then, ~5 drops (20 µl/drop) of silver nitrate solution (0.2 g gelatin, 10 ml 1% formic acid, 20 ml 50% silver nitrate; Sigma-Aldrich) were added onto the cover slips under the exclusion of light, followed by incubation at room temperature for 1 h. The cells were repeatedly washed with deionized water, dehydrated with 95–100% alcohol, cleared with xylene and mounted with neutral gum (Sigma-Aldrich). Single black particles in the nucleus were counted under a light microscope (CKX41; Olympus, Tokyo, Japan) and three horizons were randomly counted.
6
Immunohistochemical Analysis of Tight Junctions
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The slices were dewaxed in water and then placed in xylene for 20 min, which were performed three times. After that, the sections were placed in 100, 95, 85, and 75% ethanol for 5 min. Slices were soaked in distilled water for 5 min. The slices were immersed in 0.01 M of citrate buffer (pH 6.0) and boiled for 20 min. After being cooled to room temperature, the slices were washed with 0.01 M of PBS (pH 7.2~7.6) for 3 min, which were performed three times. Then 1% periodic acid was added, and the slices were placed at room temperature for 10 min. Slices were washed with PBS for 3 min, which were performed three times. Diluted primary antibodies Claudin-1 (1:100, rabbit, 13050-1-AP, PTG), Occludin (1:100, rabbit, 13050-1-AP, PTG), and ZO-1 (1:100, rabbit, 13050-1-AP, PTG) were added to the slices and put at 4°C overnight. Pan secondary antibody was added, and slices were incubated at 37°C for 30 min. DAB (Nakasugi Golden Bridge) was added to dye slices for 5–10 min. Hematoxylin (Wellbio, China) was used to dye cell nuclei for 5–10 min, and then they were washed with distilled water. They were dehydrated in all levels of alcohol (60–100%) and transparent in xylene. The slides were mounted with neutral gum (Sigma) and then observed.
7
Immunohistochemical Evaluation of Protein Markers
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Tissue slices were immersed in 0.01 M citrate buffer (pH 6.0; Wellbio, China). The buffer was brought to a boil and cooked for 20 min before being cooled to room temperature. Tissue slices were washed with PBS (pH 7.2–7.6; China) after cooling. The samples were then incubated at 4°C overnight with diluted primary antibody. Following incubation with primary antibody, tissue slices were washed with PBS, and 50–100 μL of anti-rabbit-IgG antibody-HRP polymer (Thermo Fisher Scientific, China) was added. A working solution of 50–100 μL of the chromogenic reagent DAB (Zhongshan Jinqiao Biotechnology, China) was also added. Tissues were counterstained with hematoxylin before being mounted with neutral gum (Sigma, USA). Tissue slices were then examined under a microscope (OLYMPUS, Japan, BX43). Antibodies for IHC staining included Ki-67 (1:200, Servicebio, China), PGK1 (1:200, Proteintech, China), E-cadherin (1:200, Proteintech, China), and N-cadherin (1:200, Proteintech, China). The Ki-67 expression level was graded based on the percentage of staining. The histochemistry score (H-score) was used to determine the expression levels of PGK1, E-cadherin, and N-cadherin. H-score = ΣPi (i + 1), where i denotes the intensity score and Pi denotes the percentage of the cells.
8
Visualizing Neuroinflammatory Responses in Spinal Cord
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The sections were placed in boiling 0.01 M citric acid (pH = 6) for antigen retrieval for 15 minutes. The repaired sections were blocked with goat serum blocking solution at 4°C for 2 hours, and the following primary antibodies were added to incubate overnight at 4°C: anti-nuclear factor kappa-B (NF-κB) (1:1000, Abcam, Cambridge, UK) and anti-tumor necrosis factor-α (TNF-α) (1:1000, Abcam, Cambridge, UK). On the next day, a fluorescent secondary antibody Alexa Fluor 568 (1:400; Life Technology, USA) was added to the spinal cord tissue, and the tissues were combined for 2 hours at room temperature. Subsequently, the sections were incubated with a second primary antibody (anti-neuronal nuclei [NeuN], 1:400; Novus Biologicals) for 2 hours at room temperature and then the tissues were incubated at room temperature for 2 hours with fluorescent secondary antibody Alexa Fluor 488 (1:400; Life Technology, USA). Next, the nuclei were counterstained by staining with 4′,6-diamidino-2-phenylindole (1:1000) and sealed with a neutral gum (Sigma-Aldrich, St. Louis, Missouri, USA). Tissue staining was observed under a fluorescence microscope (Leica, Heidelberger, Germany). Neurons in the anterior horn of the spinal cord were observed to evaluate changes in the inflammatory response of neurons.
9
Matrigel-Based Cell Invasion and Migration Assay
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Matrigel (Corning) was diluted with Roswell Park Memorial Institute (RPMI)-1640 medium (Solarbio, Beijing, China) and added into the chamber dropwise. Cells suspended in RPMI-1640 medium (1 × 10 4 cells/mL) were seeded into 6-well plates, while 500 μL RPMI-1640 medium supplemented with 10% fetal bovine serum was placed in the basolateral chamber. After an incubation for 24 h at 37°C with 5% CO 2 , the cells in the basolateral chamber were washed with PBS, xed in 4% paraformaldehyde for 30 min at room temperature and stained with 0.5% crystal violet for 30 min. After being xed with neutral gum (Sigma-Aldrich Chemical Company, St Louis, MO, USA), ve visual elds were randomly selected under an inverted microscope (Eclipse Ti, Nikon, Japan) for photograph and calculating the invaded cell number.
Matrigel was not added in the cell migration assay, and the other steps were the same as the invasion assay.
Matrigel was not added in the cell migration assay, and the other steps were the same as the invasion assay.
10
Cell Invasion and Migration Assay
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Matrigel (Corning) was diluted with Roswell Park Memorial Institute (RPMI)-1640 medium (Solarbio, Beijing, China) and added into the chamber dropwise. Cells suspended in RPMI-1640 medium (1 × 10 4 cells/mL) were seeded into 6-well plates, while 500 µL RPMI-1640 medium supplemented with 10% fetal bovine serum was placed in the basolateral chamber. After an incubation for 24 h at 37 °C with 5% CO 2 , the cells in the basolateral chamber were washed with PBS, xed in 4% paraformaldehyde for 30 min at room temperature and stained with 0.5% crystal violet staining solution for 30 min. After being xed with neutral gum (Sigma-Aldrich Chemical Company, St Louis, MO, USA), ve visual elds were randomly selected under an inverted microscope (Eclipse Ti, Nikon, Japan) for photograph and calculating the invaded cell number. Matrigel was not added in the cell migration assay, and the other steps were the same as the invasion assay.
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