Tissue blocks from excised tumors were fixed with 10% formalin for 48 h at room temperature and embedded in paraffin. Then, 4-µm sections were stained with hematoxylin for 5 min and eosin for 2 min at room temperature for histological examinations and morphometric analysis. For immunohistochemical staining, the paraffin sections were first deparaffinized, and then the sections were heated in citrate buffer for antigen retrieval. Sections were then incubated with 3% hydrogen peroxide in phosphate-buffered saline (PBS) at 37°C for 5 min, and blocked with 3% bovine serum albumin (BSA) in PBS at 37°C for 1 h. Slides were incubated overnight at 4°C with primary antibodies against Bax (dilution 1:100), Bcl-2 (dilution 1:400; product no. 15071; Cell Signaling Technology, Inc.), E-cadherin (dilution 1:400), N-cadherin (dilution 1:125; product no. 13116; Cell Signaling Technology, Inc.), vimentin (dilution 1:100), MMP-2 (dilution 1:100), MMP-9 (dilution 1:100) and Ki67 (dilution 1:500; product no. 9449; Cell Signaling Technology, Inc.), followed by incubation with biotinylated secondary antibodies (dilution 1:50; product nos. 8114 and 8125; Cell Signaling Technology, Inc.) for 1 h at room temperature.
Biotinylated secondary antibodies
Manufactured by Cell Signaling Technology
Sourced in United States
Biotinylated secondary antibodies are designed to detect and amplify the signal from primary antibodies in various immunoassay techniques. They bind to the primary antibody and provide a platform for the attachment of streptavidin-conjugated detection agents, enabling signal amplification and improved sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Blocking buffer, by Thermo Fisher Scientific (2 mentions)
Sav hrp conjugates, by Cell Signaling Technology (2 mentions)
Dab substrate solution, by Cell Signaling Technology (2 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Dmi3000b, by Leica (1 mentions)
Ipp software, by Media Cybernetics (1 mentions)
Polyclonal anti vegf antibody, by Abcam (1 mentions)
Mouse monoclonal anti dnmt1 antibody, by Abcam (1 mentions)
Permount, by Merck Group (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Hematoxylin, by Cell Signaling Technology (1 mentions)
Fgfr2, by Cell Signaling Technology (1 mentions)
Igf 1r, by Cell Signaling Technology (1 mentions)
Vegfa, by Cell Signaling Technology (1 mentions)
Human pdgf bb, by Merck Group (1 mentions)
7 protocols using biotinylated secondary antibodies
1
Immunohistochemical Analysis of Tumor Markers
2
Immunohistochemical Analysis of Autophagy and Inflammation
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Lungs were removed and fixed in 4% paraformaldehyde overnight and then embedded in paraffin to obtain three consecutive 5-μm thick sections. Sections were de-paraffinized in xylene and rehydrated through graded alcohol, followed by rinsing in PBS. Sections were placed in EDTA-antigen retrieval buffer and then exposed to 3% H2O2 for 10 min to block endogenous peroxidase activity at room temperature. Samples were blocked with sheep serum for 15 min and incubated in LC3B, Beclin 1, and NFκB primary antibodies (Cell Signaling, Beverly, MA, USA) diluted in blocking sera (1:200) at 4 °C overnight. After incubation, slides were washed three times and incubated with biotinylated secondary antibodies (Cell Signaling, Beverly, MA, USA). A hen enzyme reaction was performed using peroxidase substrate and sections were counterstained with hematoxylin and dehydrated through graded alcohol, mounted with neutral gum and observed under an inverted phase contrast microscope (LeicaDMI3000 B; Leica, Solms, Germany) at ×400 magnification. The immunostaining intensity assessment was performed using Image-Pro Plus 6.0 software. Mean integrated optical density (IOD) was used to express the antigen spot color intensity. The nuclear NF-κB staining was performed by counting staining nucleus. Each group contained three sections from different mice.
3
Quantitative Immunohistochemical Analysis of Aortic Arch
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Aortic arch samples fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into consecutive 8 μm thick free-floating sections were incubated with 4% bovine serum albumin (BSA) in PBS for 1 h and then reacted with one of the following antibodies: a rabbit polyclonal anti-MCP1 antibody, 1:200 (Abcam, Cambridge, UK); a rabbit polyclonal anti-VEGF antibody, 1:200 (Abcam); a mouse monoclonal anti-DNMT1 antibody, 1:500 (Abcam); a rabbit polyclonal anti-DNMT3A antibody, 1:300 (Abcam); or a rabbit polyclonal anti-DNMT3B antibody, 1:200 (Abcam) at 4 °C overnight. The sections were washed with PBS and reacted with the appropriate biotinylated secondary antibodies, 1:1000 (Cell Signaling Technology, Danvers, MA, USA) in PBS and visualized using an SABC Elite kit (BosterBio Technology., LTD., Wuhan, China). Quantitation of immunoreactive protein was performed on a random five sections and the images were obtained using a microscope (Olympus). The integrated optical density of each was determined using IPP software (Media Cybernetics, Inc., Silver Spring, MD, USA).
4
Immunostaining of Engrafted Kidneys
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The engrafted kidneys were harvested from Foxp3-Mettl14f/+ cKO mice and littermate controls after transplantation by nephrectomy. The engrafted kidney was preserved in 10% formalin overnight. Immunostaining procedures were performed using a standard approach (21 (link)). Briefly, 4 to 6 μm tissue sections were deparaffinized and washed three times with PBS. These sections were sealed with blocking buffer (Thermo Fisher Scientific, USA). Then, primary antibodies against CD4+ and CD8+ (1:200, Abcam, USA) were applied to these sections and incubated in a humidified chamber at room temperature for 2 hours. Next, these sections were incubated with Sav-HRP conjugates (Cell Signaling Technology, USA) at room temperature for 30 min in the dark. Afterward, these sections were incubated with biotinylated secondary antibodies (Cell Signaling Technology, USA) at room temperature for 1 hour. These sections were dehydrated with 95% ethanol and 100% ethanol. Next, a DAB substrate solution (Cell Signaling Technology, USA) was added to these sections to reveal the color of IHC staining. Finally, images of these sections were obtained under a microscope and analyzed with Image Pro Plus software. This assay was repeated at least three times.
5
Immunohistochemical Staining of Paraffin-Embedded Lung Tissue
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Paraffin-embedded lung tissues were sectioned at 5 μm, deparaffinized, and rehydrated using graded ethanol washes. Sodium citrate buffer was used for antigen retrieval. Sections were blocked with BSA then incubated with primary antibodies overnight. Anti-mApple antibodies were from Abcam (Cambridge, UK) and used as recommended. Biotinylated secondary antibodies were from Cell Signaling Technology and used as recommended. DAB peroxidase substrate kit from Vector Labs (Burlingame, CA) was used as recommended. Sections were then dehydrated using graded ethanol washes and mounted in Permount (Sigma). Mounted sections were imaged by light microscopy.
6
Immunohistochemical Analysis of Islet Grafts
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The rinsed section of islet grafts was immersed into 35 H2O2 (Sigma, USA) for 10 min at RT to block endogenous peroxidase activity. After washing the sections three times with PBS, these sections were sealed with blocking buffer (ThermoFisher Scientific, USA), and then the primary antibodies of IGF1R, FGFR2 or VEGFA (Cell Signaling Technology, USA) were applied to these sections and incubated in a humidified chamber at RT for two h. These sections were incubated with biotinylated secondary antibodies (Cell Signaling Technology, USA) at RT for 1 h after washing as usual. Then, these sections were incubated with Sav-HRP conjugates (Cell Signaling Technology, USA) at RT for 30 min in the darkness. Next, DAB substrate solution (Cell Signaling Technology, USA) was added to these sections to reveal the color of IHC staining. Then, these sections were counterstained with hematoxylin (Cell Signaling Technology, USA) for 1 min. After rinsing with the running tap water, these sections were dehydrated with 95% ethanol, 100% ethanol as previously mentioned. Finally, the images of these section were obtained under microscopy and analyzed with Image J software. This assay was repeated at least three times.
7
Investigating Melanoma Cell Signaling
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B16F10 melanoma cells were purchased from American Type Culture Collection (Rockville, MD, USA). PAK1-SureSilencing plasmids, attractene transfection, endofree ® plasmid maxi kit were purchased from Qiagen (Valencia, CA 91355, USA). Primary PAK1-antibodies, biotinylated secondary antibodies, signalfire TM ECL reagent were obtained from Cell Signalling Technology (Danver, MA, USA). Kinase Glo reagent and ATP (ATP_Glo kinase kit) were purchased from Promega (Madison, Wisconsin, USA). Lipofectamin and JM109 competent cells were purchased from Invitrogen and Life Technology. AG1295 and AG1478 were purchased from Calbiochem (San Diego, CA, USA). All other chemicals including human PDGF-bb were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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