The placenta tissues were embedded in paraffin and sectioned into 4‐μm slides. Then, slides were deparaffinized and hydrated. After inactivating endogenous peroxidase in 3% hydrogen peroxide, slides were retrieved in citric acid buffer (pH 6.0) by microwaving for 15 minutes. Slides were blocked in normal goat serum (Invitrogen) for 30 minutes at room temperature and incubated with an anti‐Ki67 antibody (Abcam) overnight at 4°C. Samples were then washed with TBST (Invitrogen) and incubated with the appropriate secondary antibody (Abcam) for 2 hours at 37°C. Sections were then washed with TBST and stained using a DAB Detection Kit (Solarbio, Beijing, China). Finally, sections were counterstained with hematoxylin. Section images were captured and analysed using Motic Images Advanced 3.2 software (Motic, XiaMen, China).
Images advanced 3
Manufactured by Motic
Sourced in China, Hong Kong
Motic Images Advanced 3.2 is a software application designed for image capture, processing, and analysis. It provides a comprehensive suite of tools for users to perform various imaging tasks in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Dab detection kit, by Solarbio (2 mentions)
Appropriate secondary antibody, by Abcam (1 mentions)
Anti ki67 antibody, by Abcam (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Tbs t, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
E800 microscope, by Nikon (1 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
Microtome, by Thermo Fisher Scientific (1 mentions)
Ba 410 microscope, by Motic (1 mentions)
Ba200, by Motic (1 mentions)
Ba410digital, by Motic (1 mentions)
24 protocols using images advanced 3
1
Immunohistochemical Analysis of Placental Ki67
2
Wound Healing Assay for Cell Migration
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Lines were drawn behind the 6-well plate (0.5∼1 cm/per line). Transfected cells (3 × 104) were inoculated into 6-well plates overnight. When the transfection cells reached 80%∼90% confluence, scratching was performed with the lines perpendicular to the lines on the back of the plates. Eight fields were randomly selected from each well after 48 h of incubation, and the cell movements in the scratch were observed and photographed. The relative width of the scratch was detected with Motic Images Advanced 3.2 software, and cell migration ability was examined. The experiment was repeated at least three times.
3
Histological Analysis of Liver Tissue
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Liver tissues were fixed in 10% neutral-buffered formalin for 1 week at 25–27°C, dehydrated in a 70–100% gradient of ethyl alcohol, washed in xylene, embedded in paraffin and cut into 5-µm sections. The liver sections were deparaffinized in xylene, rehydrated in a reverse-gradient series of ethyl alcohol, and stained with hematoxylin for 5 min and eosin for 5 min (H&E; Merck KGaA, Darmstadt, Germany) at room temperature. Pathological changes were observed under a light microscope (magnification, −200), and analyzed with Motic Images Advanced 3.2 software (Motic China Group Co., Ltd.).
4
Wound Healing Assay in Cell Lines
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GEO or HCT116 cells were centrifuged, and the cell suspension was cultured in six‐well plates for 24 h. After the degree of cell fusion reached 80–90%, the transfer gun was used to draw some scratches on the back of each plate with the same force. Then the plates were washed thrice with PBS (Thermo Fisher Scientific). Images were photographed at 0 and 24 h on motic images advanced 3.2 software (Motic Asia, Hong Kong, China).
5
Histological Analysis of Ram Muscle Fibers
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A total of 32 rams were randomly selected from the population of 76 to conduct histological detection at Chengdu Lilai Biotechnology Co., Ltd. The methods were referenced by Cui et al. (31 (link)), with some modifications. For haematoxylin and eosin staining (HE staining), longissimus dorsi samples were fixed overnight in 10% neutral formaldehyde solution, embedded in paraffin, and then cut into 5 μm sections. The sections were further stained according to a standard protocol and examined with a BA210Digital light microscope (Motic China Group Co., Ltd., Xiamen, China). The fiber number in each sample was manually counted in three different fields (400 × mirror), and the average was calculated. Muscle fiber diameter and perimysium thickness were measured with Motic Images Advanced 3.2 software.
6
Intestinal Villus and Crypt Morphometry
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Observation of villus height (VH) and crypt depth (CD) of the intestinal villi was conducted under a light microscope. The jejunum tissues were fixed, embedded, sectioned, and stained with hematoxylin/eosin for tissue histology. Images were processed using Motic Images Advanced 3.2 software, followed by measurements of the VH (measured from the tip to the crypt–villus junction) and CD (measured from the crypt–villus junction to the base). At least four measurements of VH or CD per explant were made.
7
Immunohistochemical Analysis of Steroid Receptors
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The cells were dispersed at a density of 1×105/ml and seeded in 12-well plates for culturing. The medium was removed subsequent to 12 days of adherent growth. The slides were fixed with 10% formalin for 60 min and endogenous peroxidase was quenched with hydrogen peroxide in methanol solution (fresh, 30% H2O2; methyl alcohol, dilution, 1:50) for 30 min. The cells were washed with distilled water and immersed in 5% bovine serum albumin for 20 min at room temperature to block the non-specific binding sites prior to incubation with the anti-androgen receptor, anti-ERα and anti-ERβ primary antibodies (dilution, 1:100) in a wet box at 4°C overnight. Biotin-labeled Goat Anti-Rabbit IgG and SABC were added successively at 37°C for 20 min, followed by staining with a DAB kit. The sections were counterstained with hematoxylin and dehydrated, washed, mounted, and observed under a Motic inverted microscope. Finally, 120 cells in each group were randomly selected to obtain the mean values with the Motic Images Advanced 3.2 software (Motic, Kowloon, Hong Kong).
8
Intestinal Morphology Analysis Protocol
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The intestinal morphology was analyzed according to our previous study (20 (link)). Briefly, the fixed intestinal samples were dehydrated and then embedded in paraffin. After that, the embedded samples were sectioned and stained with hematoxylin and eosin. The villi height (VH) and crypt depth (CD) were measured using Motic Images Advanced 3.2 software (Motic, Xiamen, China). At least 10-well-oriented intact villis and their associated crypts were analyzed in each intestinal section of each piglet. The CD was divided by VH to calculate the VH/CD ratio.
9
Immunohistochemical Analysis of Placental Proteins
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The placenta tissues were embedded in paraffin and cut into 4 μm slides. The slides were routinely deparaffinized and hydrated. After inactivating endogenous peroxidase in 3% hydrogen peroxide, slides were then retrieved in citric acid buffer (pH6.0) by microwave for 15 min. Slices were blocked in normal goat serum for 30 min at room temperature, and incubated with primary antibody overnight at 4°C. The slides were then washed with TBST and incubated with appropriate secondary antibody for 2 h at 37°C. The sections were then washed with TBST and stained by using DAB Detection Kit (Solarbio, Beijing, China). Finally, the sections were counterstained with hematoxylin. The slices were captured and analyzed using Motic Images Advanced 3.2 software (Motic, XiaMen, China). The average optical density analysis was conducted by a researcher blind to treatment conditions. Data was acquired from eight sections/animal and the data were averaged to produce the relative expression of proteins.
10
Histological Analysis of Testis Tissue
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Fixed samples were washed with 70–100% (v/v) ethanol, embedded in paraffin, sliced into 5 µm thick sections using a microtome (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and mounted on glass slides. The mounted tissues were subsequently rehydrated with xylene and 100% ethanol and stained with hematoxylin and eosin (Sigma-Aldrich). Representative images of stained testes sections were obtained using a Nikon E-800 microscope (Nikon E-800, Tokyo, Japan) and Motic Images Advanced 3.2 software (Kowloon, Hong Kong).
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