Briefly, mice were killed with overdose CO2, eyes were embedded in Tissue Freezing Medium (Sakura Tissue-Tek O.C.T) at 4°C for 10 min, -80°C for 30 min, and sectioned at 10 μm in a cryostat. Sections were fixed with 4% paraformaldehyde (PFA) for 15 min, washed with PBS. HE staining (Solarbio) was used to observe the structure of the retina. Sections were examined with a LEICA DM1000 microscope. 10× and 40× objective was used for histological measurements. The images were captured by LAS V48 system and were then further processed with Image J. Twelve tissue blocks from six mice (two blocks per mouse) were taken from each group. Three areas of the retina (central, media, periphery) were included in the measurements (2∼3 measurements per tissue bloc). The following parameters were measured: (i) The thickness of retinal cross-section from outer limiting membrane to the inner limiting membrane (OLM-ILM); (ii) the width of each layer of retina.
H e staining
Manufactured by Solarbio
Sourced in China
H&E staining is a standard histological staining technique used to visualize the cellular structure of tissues. It involves the use of two dyes, hematoxylin and eosin, which stain the cell nuclei blue and the cytoplasm and extracellular matrix pink, respectively. This staining method allows for the differentiation of various cell types and tissue components within a sample.
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Lab products found in correlation
Formic acid, by Thermo Fisher Scientific (3 mentions)
Masson s trichrome staining, by Solarbio (2 mentions)
Inverted microscope, by Leica (2 mentions)
Mouse anti human chn1 monoclonal antibody, by Abcam (2 mentions)
Rabbit anti human snail polyclonal antibody, by Proteintech (2 mentions)
Pv 6002 kit, by ZSGB-BIO (2 mentions)
Neutral paraformaldehyde, by Biosharp (2 mentions)
Bx51 microscope, by Olympus (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Dp71 microscope, by Olympus (1 mentions)
Lh m100cb, by Nikon (1 mentions)
G1340, by Solarbio (1 mentions)
G1120, by Solarbio (1 mentions)
Methyl green solution, by Merck Group (1 mentions)
Trap staining solution, by Merck Group (1 mentions)
Dm3000, by Leica (1 mentions)
Rm2235, by Leica (1 mentions)
Abc hrp kit, by Thermo Fisher Scientific (1 mentions)
Masson s trichrome mt staining, by Solarbio (1 mentions)
Collagen 1, by Abcam (1 mentions)
35 protocols using h e staining
1
Histological Analysis of Murine Retina
2
Histological Evaluation of Neovascularization
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At POD 7, the animals were euthanized, and six samples (0.5 cm×0.5 cm) in the middle tissue of area II were harvested. Histologic examination was performed on these tissue samples, which were fixed in 4% neutral buffered formalin for 24 hrs, embedded in paraffin, sliced into 4 μm thickness, and subsequently stained with H&E staining (Solarbio Science & Technology, Beijing, People's
Republic of China). Under a light microscope (200× magnification), neovascularization, edema, and thickness of granulation tissue in flaps were evaluated. To assess angiogenesis level, mean vessel density (MVD) was counted manually by the number of microvessels per unit area (/mm2). Six random fields from three random sections of each tissue sample were determined for counting.
Republic of China). Under a light microscope (200× magnification), neovascularization, edema, and thickness of granulation tissue in flaps were evaluated. To assess angiogenesis level, mean vessel density (MVD) was counted manually by the number of microvessels per unit area (/mm2). Six random fields from three random sections of each tissue sample were determined for counting.
3
Histological Analysis of Pancreatic and Liver Injury
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Pancreatic and liver tissues were collected, fixed immediately in 4% paraformaldehyde for 24 h, dehydrated in a graded ethanol series, and then embedded in paraffin. The tissue blocks were cut into 4.5 μm-thick sections, dewaxed, and hydrated. Then, pancreatic and liver sections were stained with hematoxylin and eosin (H&E) staining (G1120, Solarbio, Beijing, China) according to the manufacturer’s instructions. After observation under an Olympus BX-51 microscope (Olympus Corporation, Tokyo, Japan), histological scores were obtained to evaluate the degree of pancreatic and liver injury with Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, United States) as described elsewhere (Rongione et al., 1997 (link); Elshal et al., 2019 (link)).
4
Quantifying Trabecular Bone Differences
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In order to compare the differences of trabecular bone among three groups, H&E staining was performed. Briefly, after decalcified in EDTA, samples were dehydrated through a graded alcohol series and then embedded in paraffin. Serial sections with a thickness of 4 μm were cut and mounted on polylysinecoated microscope slides. H&E staining (Solarbio, Shanghai, China) was performed subsequently according to manufacturer’s protocol and finally an examination under a microscopic light by using an Olympus DP71 microscope (Olympus Co., Japan) was held. We calculated the mean values of the percentage of trabecular bone in five observation points (Fig. 1 c) with ImageJ (version 1.8.0) and compared them in histograms.
5
Histological Analysis of Murine Tissues
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At the end of animal experiments, the tissues including tumor, heart, liver, spleen, lung, and kidney were excised from the mice, fixed in 4% paraformaldehyde at room temperature, gradually dehydrated by successively immerging the specimens in 70% (overnight), 80% (~ 4 h), 90% (~ 1 h), and 100% (~ 1 h) ethanol, embedded in paraffin, and sectioned to slides with a thickness of 5 μm. After deparaffinizing with xylene (Tianjin Damao Chemical Reagent Factory, Tianjin, China), the tissue slides were hydrated with ethanol and water, stained with hematoxylin and eosin (i.e. H&E staining; both were from Solarbio Science & Technology Co.), dehydrated, mounted on glass cover slides, and images by an inverted microscope (Nikon LH-M100CB, Japan).
6
Maxillary Molar Histomorphometry in Orthodontics
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After micro-CT scanning, the left half of the maxilla of each animal was decalcified in 14% ethylenediaminetetraacetic acid (EDTA) (pH 7.4) for a month. Then, all the specimens were dehydrated in a series of alcohol baths and embedded in paraffin. Next, the samples, including the maxillary molars, were excised into 5 μm thick frontal sections in the sagittal direction. H&E staining (G1120; Solarbio, Beijing, China), tartrate-resistant acid phosphatase (TRAP) staining (Sigma-Aldrich, St. Louis, MO, United States), and Masson’s trichrome staining (G1340; Solarbio, Beijing, China) were performed for the histological analyses. Multinucleated cells adjacent to the tension side of the periodontal area were counted as TRAP-positive osteoclasts. Two independent investigators counted the number of TRAP-positive cells. Masson’s trichrome staining was used to identify the collagen fiber arrangement on the tension side.
7
Immunohistochemical Analysis of CHN1 and Snail
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The paraffin-embedded sections were de-waxed, hydrated, and antigen retrieved in 0.01 M citrate solution at 95 °C for 20 min. They were then treated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Mouse anti-human CHN1 monoclonal antibody (1:150, Abcam, UK) and rabbit anti-human Snail polyclonal antibody (1:500, Proteintech Group, USA) were used for incubation, followed by serum blocking. A PV-6002 kit (ZSGB-Bio, Beijing, China) was used in the following immunodetection. The specimens were observed and captured using an inverted microscope (Leica, Wetzlar, Germany). Tumors harvested from the xenograft nude mice were fixed and embedded in paraffin, and their pathological features were analyzed by H&E staining (Solarbio Life Science, Beijing, China).
8
Mouse Femur Histological Analysis
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The mouse femurs were fixed and decalcified, and then embedded in paraffin. Sections of 4.5 μm were prepared. H&E staining (Solarbio, Beijing, China) was performed as the manufacturer’s instruction. The stained sections were observed using an inverted microscope (IX81, Olympus).
TRAP staining was performed according to the procedure described previously (Liu et al., 2016 (link)). TRAP staining solution (Sigma) was added to cover the tissue sections. After incubating at 37°C for 40 min, the sections were stained with 0.1% methyl green solution (Sigma) for 10 s. The stained sections were observed using an inverted microscope (IX81, Olympus).
TRAP staining was performed according to the procedure described previously (Liu et al., 2016 (link)). TRAP staining solution (Sigma) was added to cover the tissue sections. After incubating at 37°C for 40 min, the sections were stained with 0.1% methyl green solution (Sigma) for 10 s. The stained sections were observed using an inverted microscope (IX81, Olympus).
9
Histological Analysis of Lung Tumors
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After sacrificing the mice, samples were fixed in 4% paraformaldehyde overnight and then embedded in paraffin. Paraffin sections were cut into 3 μm on a slicer (Leica RM2235, German). After dewaxing in xylene and gradient alcohol, the sections were then processed for H&E staining (Solarbio, China). Pictures were taken at a 20x (H&E) magnification by using a microscope (Leica DM3000, German). For the immunohistochemistry of CD8 in lung tumour tissues, antigen retrieval was performed by using citric acid and sodium citrate. Then the sections were incubated with CD8 (1:500, Abcam, USA) at 4 °C overnight and followed by signal amplification using a ABC HRP Kit (Thermo, USA). Microscope (Leica, German) was used to visualize the sections.
10
Histological Analysis of Skin Samples
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Skin samples were 1 cm × 1 cm × 0.15 cm, and were fixed in 0.01 M PBS (pH 7.4) containing 4% paraformaldehyde for 8 h and then routinely processed to wax. Sections were cut at 5 μm and adhered to poly-l -lysine coated glass slides. The histology of subcutaneous tissues was analyzed with H&E staining (Solarbio, Beijing, China), or with Masson’s trichrome staining (Solarbio, Beijing, China) for the evaluation of collagen content in healing wounds.
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