Tissue microarrays (TMAs) containing 80 pairs of HCC and matched paracancerous tissues were purchased from shanghai Outdo biotechnology company Ltd. (HLivH160CS02, Shanghai, China). Immunohistochemistry (IHC) staining of CXCL2 was conducted using a Histomouse SP Kit (959551, Invitrogen, USA) according to the manufacturer’s protocal. The concentration of antibody against CXCL2 was 1:100. The results of CXCL2 staining in tissues were independently evaluated by two pathologists. The evaluation of proportion score was on a scale of 1-4 (1, 0%-25%; 2, 25.1%-50%; 3, 50.1%-75%; 4, 75.1%-100%). The staining intensity score was graded as follows: 0, negative; 1, weak; 2, moderate; 3, strong. Then the histologic score for each tissue was calculated with the formula: histologic score = proportion score × intensity score.
Histomouse sp kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HistoMouse-SP kit is a laboratory product designed for the detection of mouse antigens in histological samples. It provides a simple and effective method for identifying the presence of mouse-derived proteins in tissue sections.
Automatically generated - may contain errors
Lab products found in correlation
Dab substrate kit, by Vector Laboratories (4 mentions)
Pt link pre treatment module tissue processor, by Agilent Technologies (2 mentions)
Anti insulin, by Abcam (2 mentions)
Anti f4 80 antibodies, by Merck Group (2 mentions)
Mac 3, by BD (1 mentions)
Mcp 1, by Novus Biologicals (1 mentions)
Ab183316, by Abcam (1 mentions)
Ab185444, by Abcam (1 mentions)
9 protocols using histomouse sp kit
1
Immunohistochemical Analysis of CXCL2 in HCC
2
Paraffin Embedding and Immunohistochemistry of Embryonic Hearts
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Embryonic hearts were fixed in 4% paraformaldehyde for 30 min at room temperature and embedded in paraffin. Four µm sections were cut for hematoxylin and eosin or immunohistochemical staining as previously described 20 (link). Antigen retrieval was conducted in EDTA buffer (pH: 9.0) by heating to 99°C for 20 minutes with a DAKO PT Link Pre-Treatment Module Tissue Processor (Carpinteria, CA). Primary antibodies (Table S4 ) were applied to slides for overnight at 4°C. Immunohistochemical staining was performed using HistoMouse™-SP Kit (Invitrogen, Carlsbad, CA) according to manufacturer’s instruction throughout this study.
3
Immunohistochemical Analysis of OC Tissue
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The OC tissue array (Cat NO: HOvaC154Su01) was acquired from Shanghai Outdo Biotech. The related clinicopathological details were also offered by this company. Immunohistochemical (IHC) was conducted as previously depicted [38 (link)]. Briefly, IHC was accomplished using Histomouse SP Kit (Invitrogen, USA). Sections were immunostained utilizing a streptavidin-peroxidase method following microwave antigen retrieval. 3,3′-diaminobenzidine was adopted to visualize positive signals. The antibodies against NR2F1 (Cat NO: 24573-1-AP) and platelet-derived growth factor receptor alpha (PDGFRA) (Cat NO: bs-0231R) were purchased from Proteintech. Two pathologists were invited independently to review and quantify the image of sections. IHC intensity score was subjectively ranked into four levels: 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). Scores for staining extent were assigned as: 0 (≤10%), 1 (11–25%), 2 (26–50%), 3 (51–75%), or 4 (>75%). We calculated the final score through themultiplication of the two above-mentioned scores. Eventually, total scores > 1 were grouped as high expression, while those < 1 were classified as low expression.
4
Histological Analysis of Vascular Remodeling
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Paraffin-embedded cross sections (5 µm) were stained with haematoxylin and eosin (H&E), Verhoeff-van Gieson (VVG), or Alizarin red (Ricca Chemical Company), MPO (Abcam), Mac-3 (BD Pharmingen), MCP-1 (Novus Biologicals), and matrix metalloproteinase (MMP)-2/MMP-9 (Calbiochem) antibodies were used with the HistoMouse-SP kit (Invitrogen) or DAB Substrate kit (Vector Labs). The number of elastin breaks was counted in three sections per mouse to quantify elastin degradation.26 Vascular calcification was detected by Alizarin Red staining. To quantify the immunostaining data, total stained area in the media and adventitial region was analysed using Image-Pro Plus Software.27
5
Aorta Histology and Protein Expression
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Paraffin-embedded cross sections of aortas were used for hematoxylin and eosin (H&E), Verhoeff van Gieson (VVG) and immunostaining staining for Mac-3, matrix metalloproteinase (MMP)-2 and−9. Antibodies for Mac-3 (BD Pharmingen) and MMP-2/-9 (Calbiochem) were used with the HistoMouse-SP kit (Invitrogen) or DAB Substrate kit (Vector Labs).
6
Paraffin Embedding and Immunohistochemistry of Embryonic Hearts
7
Histological Analysis of Pancreatic, Liver, and Adipose Tissues
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Pancreas, liver and adipose tissues were fixed by immersion in neutral buffered formalin (10%), dehydrated in ethanol and then transferred to xylene solution for embedding in paraffin. Five μm sections were stained with hematoxylin-eosin (H&E) or incubated with anti-insulin (Abcam, 1:100 dilution, to detect beta cells) or anti-F4/80 antibodies (Sigma-Aldrich, 1:100 dilution, to detect macrophages) for 4 h at 37 °C, and then processed with HistoMouse-SP kit (Invitrogen) or DAB Substrate kits (Vector Labs) according to the manufacturers’ protocols. The number of F4/80-positive crown-like structures were counted in 5 randomly selected high-power fields and normalized to mm2 for quantification.
8
Histological Analysis of Pancreatic, Liver, and Adipose Tissues
9
Quantitative Immunohistochemistry of Glioma
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The glioma and normal brain tissues were obtained from the Department of Pathology, Xiangya Hospital. The tissues were paraffin embedded and the immunohistochemistry (IHC) was performed as previously described (29 (link), 30 (link)). In brief, IHC was conducted using a Histomouse SP Kit (959551; Invitrogen, Waltham, MA, USA). Paraffin sections were immunostained through a streptavidin peroxidase procedure after microwave antigen retrieval. The signal was detected using a 3,3′-diaminobenzidine solution. The antibodies against MXRA8 (1:100, ab185444) and CSF1R (1:100, ab183316) were all purchased from Abcam. Images of the sections were independently examined and differentially quantified by two pathologists. IHC intensity score was scored as 0 (negative), 1 (weak brown), 2 (moderate brown), or 3 (strong brown). The extent of staining was scored as 0 (≤10%), 1 (11%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (>75%). The final staining score was determined by multiplication of intensity scores and extent score and was classified as weakly positive (1–3), positive (4–6), and strongly positive (7–12). All paraffin-embedded specimens were collected following the ethical standards of the human experimental committee.
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