BALB/c nude mice (5 weeks old, male) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. After 3 days of acclimatization, mice were injected subcutaneously (s.c.) with 1.0 × 106 iPS cells (passage 5) in the posterior flanks. Mice were maintained for 4 weeks before the animals were humanely sacrificed and the teratomas excised, fixed, and subjected to histopathological analysis using H&E staining (Servicebio). Studies were conducted with approval from the Animal Research Ethics Committee of the University of Science and Technology of China.
H e staining
Manufactured by Wuhan Servicebio Technology
Sourced in China
H&E staining is a standard histological technique used to stain tissue samples. It involves the use of two dyes: hematoxylin, which stains nuclei blue, and eosin, which stains cytoplasm and other structures pink or red. This staining method allows for the visualization of cellular and tissue morphology under a microscope.
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Lab products found in correlation
Masson s trichrome staining, by Wuhan Servicebio Technology (4 mentions)
Confocal fluorescence microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
Dmi8 m, by Leica (1 mentions)
α sma, by Abcam (1 mentions)
Nrp 1, by Thermo Fisher Scientific (1 mentions)
Masson staining, by Wuhan Servicebio Technology (1 mentions)
Vegfr2, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Wuhan Servicebio Technology (1 mentions)
Triton x 100, by Wuhan Servicebio Technology (1 mentions)
Ae1 ae3, by Abcam (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Comprehensive laboratory animal monitoring system, by Columbus Instruments (1 mentions)
Insulin, by Roche (1 mentions)
Rpmi 1640 medium, by Sartorius (1 mentions)
Ecl substrate, by Merck Group (1 mentions)
Sds loading buffer, by Beyotime (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
Il 27rα mab, by R&D Systems (1 mentions)
Antibody dilution buffer, by Beyotime (1 mentions)
19 protocols using h e staining
1
Teratoma Formation Assay in BALB/c Nude Mice
2
Comprehensive Aortic Sinus and Plaque Analysis
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For aortic sinus analysis, hearts were fixed in 4% formalin, dehydrated, embedded with paraffin, and sliced into 5-μm-thick sections from the aortic region towards the apex of the heart. H&E staining (Servicebio) was then performed. For plaque analysis, fibrous caps and necrotic cores were examined using Masson’s trichrome staining (Servicebio). To measure fibrous cap thickness, at least three measurements of the thinnest fibrous cap within one atherosclerotic plaque were taken and averaged, as described previously.14 (link) The necrotic core area was analyzed by measuring the total acellular area in atherosclerotic plaque, as described previously.14 (link) F4/80 staining was performed to determine the macrophage content in the atherosclerotic plaque. Sections were stained with anti-F4/80 antibody (1:500, rat anti-mouse monoclonal antibody) followed by incubation with goat anti-rabbit (1:300) antibody. Slides were mounted with mounting medium containing DAPI (Servicebio). Images were analyzed in the area of atherosclerotic lesions using a confocal fluorescence microscope (Olympus, Tokyo, Japan) and Image-Pro Plus software (Media Cybernatics).
3
Liver Tissue Preparation for Histology
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Apportion of freshly extracted liver tissue was cut into ∼3-mm slices and washed in PBS at RT, fixed in 4% w/v paraformaldehyde for 10 min at RT, and then incubated for 6 h at 4 °C. After a PBS rinse, the samples were dehydrated in 80%, 90%, 95%, and 100% ethanol solutions for 1 h each at 4 °C and finally in dimethyl benzene at RT. Paraffin immersion was at 60 °C for 2 h and then embedding at 4 °C. A rotary microtome was used for cutting 5-μm sections at 4 °C. The sections were then deparaffinized and prepared for H&E staining (Servicebio). Images were acquired under a Leica microscope (Leica DMi8-M).
4
Subcutaneous and Intravenous Tumor Models
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For subcutaneous inoculation, 5 × 105 B16 cells were injected at the right flank of 6–8-week-old female C57BL/6 mice. Tumor volume, which was calculated as (length × width2)/2, was recorded at different time points. For intravenous implantation, 2 × 105 B16 cells and 1–2 × 106 MDA-MB-231, A375 or MCF7 cells were injected into the lateral tail vein of 6–8-week-old female C57BL/6 mice and SCID mice, respectively. Tumor colonies in the lung were counted at day 16 for B16, at day 29 for A375 cells and at day 30 for MCF7 cells. The tumors formed by MDA-MB-231 cells were monitored by bioluminescence imaging. Briefly, D-luciferin was intraperitoneally administrated at 200 mg/kg at various time points. Fifteen minutes after injection, mice were anesthetized and bioluminescence was imaged with a charge-coupled device camera (IVIS; Xenogen). MDA-MB-231 tumors in the lung were also examined by H&E staining, which was entrusted to Servicebio (Wuhan, China).
5
Histopathological Analysis of Murine Lungs
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Mice were euthanized by an intraperitoneal injection of sodium pentobarbital (100 mg/kg), and lung tissues were carefully removed. Hematoxylin and eosin (H&E) staining was performed to observe the pathological changes of the lung. Lung tissues were obtained from the mice of each group and fixed using 4% formaldehyde in PBS in 4˚C overnight, after which tissues were dehydrated using a Leica TP 1020 Rapid Tissue Processor (Leica, Inc.). Samples were then embedded in paraffin and serial sections (4 µm thick) were stained H&E staining (Servicebio, Inc.) in room temperature. All experiments were performed according to the manufacturers' instructions. To observe the morphology of lung tissues, slides were evaluated under a Leica photograph light microscope (Leica Microsystems GmbH; magnification, x200).
6
Kidney Weight and Histology Analysis
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Twenty-four hours after the last treatment administration, the body weights of all mice were measured. Then, the mice were sacrificed, and the kidneys were excised from each mouse. Organ coefficients were calculated as the organ wet weight percentage of the total body weight. Pathological changes in kidneys tissues from 3 randomly selected mice from each group were examined using HE staining (Servicebio, Wuhan China).
7
Histological Evaluation of Liver Tissue
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HE staining (Servicebio) was performed on thin slices that were embedded in a wax block of liver tissue. The whole dyeing process includes five contents: dewaxing, dyeing, dehydration, transparent, and mounting. The overall characteristics of the tissue were observed under a low‐power microscope, and then the images of representative areas were observed and collected.
8
Histological Analysis of Colon Tissue
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Biopsy tissues were fixed with 4% paraformaldehyde for 48 h and covered with paraffin for sectioning into 4 μm sections and H&E staining (Servicebio, G1003, China) for histological analysis.
IHC of colon sections from lesion sites was performed to investigate the expression of adenomatosis polyposis downregulated 1 (APCDD1). Colon tissues were immersed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned and antigen retrieval was performed by boiling slides for 20 min in ethylene diamine tetraacetic acid citrate buffer (pH = 6.0). Sections were incubated with anti-APCDD1 antibody (Signalway antibody, 43286, China, 1:50) at 4°C overnight, washed with PBS/Tween20 buffer (pH = 7.4) and anti-rabbit secondary antibody added for 50 min at room temperature. The 3,3′-diaminobenzidine chromogenic reagent kit was used to visualize staining under the microscope (SOPTOP, CX40, China), and samples were analyzed by ImageJ software (NIH, bundled with 64-bit Java 1.8.0_172).
IHC of colon sections from lesion sites was performed to investigate the expression of adenomatosis polyposis downregulated 1 (APCDD1). Colon tissues were immersed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned and antigen retrieval was performed by boiling slides for 20 min in ethylene diamine tetraacetic acid citrate buffer (pH = 6.0). Sections were incubated with anti-APCDD1 antibody (Signalway antibody, 43286, China, 1:50) at 4°C overnight, washed with PBS/Tween20 buffer (pH = 7.4) and anti-rabbit secondary antibody added for 50 min at room temperature. The 3,3′-diaminobenzidine chromogenic reagent kit was used to visualize staining under the microscope (SOPTOP, CX40, China), and samples were analyzed by ImageJ software (NIH, bundled with 64-bit Java 1.8.0_172).
9
Immunohistochemical Analysis of Liver Fibrosis
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Immunohistochemistry was performed as previously described [18 (link)]. Liver sections were fixed with formalin and embedded in 5-μm-thick paraffin sections. Liver specimens were incubated with antibodies against NRP-1 (Invitrogen, Carlsbad, CA, USA), VEGFR2 (Cell Signalling Technology, Boston, MA, USA), CD31, and α-SMA (Abcam, Cambridge, UK). H&E staining was used to visualise histopathological structures (Servicebio, Wuhan, CN). Masson staining depicted the fibrotic strands in blue, and the intensity of the colour was associated with the degree of fibrosis (Servicebio, Wuhan, CN).
10
Histological Analysis of Skin Reepithelization
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Frozen sections or paraffin sections in groups were prepared as described above. Re-epithelialization and scar formation were assessed by H&E staining (Servicebio, China). Collagen deposition was detected by Masson’s trichrome staining (Servicebio, China). For immunohistochemical staining, the sections were blocked with 1% bovine serum albumin (Servicebio, China) and 0.5% Triton-X100 (Servicebio, China) and then separately treated with AE1/AE3 (Abcam, USA, prediluted), CD31(Abcam, USA, 1:500), CD68 (Abcam, USA, 1:500) and P63 (Abcam, USA, 1:500) mouse anti-human IgG antibody at 4 °C overnight. After washing three times with PBS for 5 min, the sections were incubated for 1 h with an HRP goat anti-mouse IgG antibody (Invitrogen, USA, 1:1000) at room temperature. Finally, the sections were stained with 3,3 N-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. The slides were covered with coverslips and examined under a microscope.
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