On day 14, we sacrificed mice and fixed hearts with 4% paraformaldehyde for 24 h. Then, we cut the paraffin-embedded sample into 5 µm thick tissue sections, dewaxed in xylene (Sangon Biotech co., Ltd., Shanghai, China), rehydrated using ethanol, stained the sections using Masson’s trichrome reagent, and finally sealed the sections using neutral gum. We calculated the wall thickness of the infarct zone and the fibrosis percentage of sections using Image J V1.53e software under an optical microscope. For each section, we took three measurements of different parts of the infarct area for thickness analysis.
Xylene
Manufactured by Sangon
Sourced in China
Xylene is a clear, colorless hydrocarbon liquid used as a solvent and a laboratory reagent. It is a mixture of three isomeric compounds: ortho-xylene, meta-xylene, and para-xylene. Xylene has a characteristic aromatic odor and is widely used in various industrial and laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Sangon (3 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Gradient ethanol, by Sangon (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Diaminobenzidine dab, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Inverted fluorescence microscope, by Nikon (1 mentions)
H e staining kit, by Sangon (1 mentions)
Formalin, by Sangon (1 mentions)
Eclipse e200 microscope, by Nikon (1 mentions)
Ab2413, by Abcam (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Dab plus substrate, by Thermo Fisher Scientific (1 mentions)
Axioskop, by Zeiss (1 mentions)
Tbst buffer, by Sangon (1 mentions)
7 protocols using xylene
1
Evaluating Cardiac Infarct Remodeling
2
Animal Tumor Histological Staining
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Animal tumor slides heated at 56°C for 1 hour, dewaxed the slide with xylene(Sangon Biotech, shanghai, China), 95% and 75% ethanol(Sangon Biotech, shanghai, China), and water; then stained the slide with hematoxylin(Sangon Biotech, shanghai, China) for 5min, washed the slides with water for 30s, dipped in 1% hydrochloric acid (Haoran Biological technology CO., LTD, shanghai, China) for 3 sec and washed with water for 10min, and stained the slide with 1% eosin (Sangon Biotech, shanghai, China) for 3min, then washed with water. Finally dehydrated, transparented and mounted the slides.
3
Myocardial Infarct Size and Wall Thickness Evaluation
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Mice were sacrificed by cervical dislocation on day 28. Then, we used Masson’s trichrome staining to determine infarct size and the thickness of the wall in the infarct zone. In brief, heart samples were fixed with 4% paraformaldehyde at room temperature for 24–48 h. Then, we embedded samples of heart tissue in paraffin and prepared tissue sections (5 µm in thickness); these were then dewaxed in xylene (Sangon Biotech Co., Ltd.) and rehydrated using ethanol. Finally, sections were stained with Masson’s trichrome reagent and sealed with neutral gum. After observation with an optical microscope, the infarct area was calculated by the sum of the endocardial and epicardial length of the infarct zone divided by the total length of the endocardial and epicardial left ventricle. In addition, the thickness of the wall in the infarct area was averaged from three to five measurements of scar thickness using Image J V1.53e software.
4
Immunohistochemical Analysis of EFNA1
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Paraffin sections were baked at 60 °C for 2 h, dewaxed with xylene (Shanghai Sangon, China), as well as gradient ethanol dehydration, and were washed by PBS, which were then repaired with high pressure antigen for 10 min, and washed PBS again. The sections were soaked in 3% H2O2 for 10 min to block exogenous peroxidase activity, closed with goat serum for 10 min (at room temperature), and then were incubated with added rabbit anti-human EFNA1 antibody (ab238505, Abcam, USA, 1:100) at 4 °C overnight. The above sections were incubated with sheep anti-rabbit II (6926-100, Emmett Technology, China, 1:1000) at room temperature for 20 min and colored with added DAB (Zhongshan Jinqiao, Beijing) for 3 min. Hematoxylin (Shanghai Sangon, China) was added for re-dying for 2 min, with gradient ethanol for dehydration and resinene for sealing, which was placed under the microscope (Olympus, Japan) for observation.
The immunopathology score was performed by 2 senior pathology physicians in our hospital, and the total result was given according to the total score of the product of dyeing area and intensity. Dyeing area: < 10% (0 point), 10–49% (1 point), 50–75% (2 points), > 75% (3 points); Dyeing intensity: uncolored (0 point), pale yellow (1 point), brown (2 points), dark brown (3 points), the total score ≥ 4 represents high expression, and the total score < 4 represents low expression.
The immunopathology score was performed by 2 senior pathology physicians in our hospital, and the total result was given according to the total score of the product of dyeing area and intensity. Dyeing area: < 10% (0 point), 10–49% (1 point), 50–75% (2 points), > 75% (3 points); Dyeing intensity: uncolored (0 point), pale yellow (1 point), brown (2 points), dark brown (3 points), the total score ≥ 4 represents high expression, and the total score < 4 represents low expression.
5
Comprehensive Histological Staining Techniques
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The HE staining kit (Sangon, Shanghai, China) and Cell Apoptosis Detection Kit (Sangon) were adopted to conduct HE and TUNEL staining by strictly following the kits' instructions. For the completion of IF and IHC staining, the sections were deparaffinated with xylene (Sangon) and rehydrated with gradient ethanol (Sangon). Then, the sections were subjected to antigen retrieval with the microwave method, and endogenous peroxidase was removed with H 2 O 2 (Sangon). After blocking with bovine serum albumin (BSA) (Beyotime), the sections were incubated with primary antibodies at 4°C overnight. Subsequently, the sections were incubated with secondary antibodies. Then, for IHC staining, diaminobenzidine (DAB) (Sigma) and hematoxylin (Sigma) were used to stain the secondary antibody and nuclei; for IF staining, the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma). Finally, the sections were observed under an inverted fluorescence microscope (Nikon, Tokyo, Japan). The antibodies are listed in Table 1.
6
Paraffin-Embedded Tissue Sectioning and Histological Staining
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The tissues, fixed in 10% formalin (Sangon Biotech, Co., Ltd.) for 24 h at room temperature, were embedded in paraffin and were cut into 4-µm sections. The sections were then deparaffinized with xylene (Sangon Biotech, Co., Ltd.) and rehydrated in a descending ethanol serials. The hematoxylin ((Sangon Biotech, Co., Ltd.) was used to stain the nuclei at room temperature for 5 min. The cytoplasm was stained with eosin (Sangon Biotech, Co., Ltd.) for 2 min at room temperature. The images were taken by a Nikon ECLIPSE E200 microscope (Nikon Corporation).
7
Immunohistochemical Fibronectin Expression Assay
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Protein expression was assayed by immunohistochemistry using the paraffin-embedded sections from the patients. The sections were first deparaffinized by xylene (Shanghai Sangon, Shanghai, China). Then, the sections were incubated with rabbit polyclonal anti-fibronectin antibody (1:500, ab2413; Abcam, Cambridge, MA, USA) at 4°C for 24 h. After washing with TBST buffer (Shanghai Sangon) for three times, the sections were incubated with goat anti-rabbit IgG H&L (HRP, 1:500, ab6721; Abcam) at room temperature for 1 h. Then, protein immunostaining was performed using DAB Plus substrate (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. The scoring of the stained tumor cells were divided into five grades: 0 (positive cells <5%), 1 (positive cells between 5 and 25%), 2 (positive cells between 26 and 50%), 3 (positive cells between 51 and 75%), and 4 (positive cells between 75 and 100%). The cells were observed using a light microscope (Axioskop; Zeiss, Germany). We designated grade 1 and 2 as low expression of fibronectin and grade 3 and 4 as high expression of fibronectin for the prognosis analysis.
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