Fluorescence immunohistochemistry was performed on paraffin sections as previously described in detail [7 (link)]. For confocal fluorescent double-labeling or triple labeling with primary antibodies from different species, antibodies were applied simultaneously at 4 °C overnight. After washing with DAKO washing buffer (DakoCytomation, Glostrup, Denmark), secondary antibodies consisting of donkey-anti-mouse Cy3 or donkey-anti-mouse Dylight 546 (Jackson ImmunoResearch, 1:200), biotinylated donkey-anti-rabbit (Amersham Pharmacia Biotech; 1:200) and (in case of triple labeling) Cy5-conjugated donkey-anti-goat, were applied simultaneously for 1 h at room temperature, followed by application of streptavidin-Cy2 (Jackson ImmunoResearch; 1:75) for 1 h at room temperature. Fluorescent preparations were then stained with 4′,6′-diamidino-2-phenylindole (DAPI, Sigma) or TO-PRO-3 (Thermo Scientific, Waltham, MA, USA) as nuclear counterstain, embedded and examined using a confocal laser scan microscope (Leica SP5, Leica Mannheim, Germany) equipped with lasers for 504, 488, 543 and 633 nm excitation. Scanning for DAPI (504 nm), Cy2 (488 nm), Cy3 (543 nm), Cy5 (633 nm) or TO-PRO-3 (633 nm) was performed sequentially to rule out fluorescence bleed through.
Dako washing buffer
Manufactured by Agilent Technologies
Sourced in Denmark
DAKO washing buffer is a laboratory solution used for washing and rinsing purposes during immunohistochemical and in-situ hybridization procedures. It is designed to maintain the integrity of biological samples and facilitate the removal of unbound reagents.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin cy2, by Jackson ImmunoResearch (1 mentions)
Sample buffer solution, by Fujifilm (1 mentions)
Pvdf blocking reagent, by Toyobo (1 mentions)
Can get signal solution 1, by Toyobo (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
Supersep, by Fujifilm (1 mentions)
P c met, by Cell Signaling Technology (1 mentions)
Las 4000, by Fujifilm (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
3 protocols using dako washing buffer
1
Fluorescent Immunohistochemistry Protocol for Confocal Microscopy
2
Western Blot Analysis of Signaling Proteins
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Treatment of the specimen was as described previously (25 (link)–27 (link)). The cell lysate was boiled in sample buffer solution (Wako). Total cell protein extracts (20 μg/lane) were separated by sodium dodecylsulface-polyacrylamide gel electrophoresis using SuperSep™ (Wako) and were electrophoretically transfected onto polyvinyl difluoride menmbranes. The membranes were blocked with PVDF blocking reagent (Toyobo, Osaka, Japan) for 1 h. The membrane was then incubated with primary antibodies against β-actin, phosphorylated type of extracellular signal-regulated kinase (p-ERK), c-jun N-terminal kinase (p-JNK), p-AKT, mammalian target of rapamycin (p-mTOR), hepatocyte growth factor receptor (p-cMet), epithelial growth factor receptor (p-EGFR) and caspase-3 (1:5,000; Cell Signaling Technology, Danvers, MA, USA) and EGFR inhibitor (1:5,000; Calbiochem 324674, Merck KGaA, Germany) overnight at 4°C. The primary antibodies were diluted with Can Get Signal Solution 1 (Toyobo). The membrane were then washed with Dako washing buffer (Dako, Glostrup, Denmark) and incubated with the appropriate secondary antibodies (1:25,000; Millipore, Darmstadt, Germany), which were diluted with Can Get Signal Solution 2. The immunoreactive proteins were visualized by chemiluminescence using a LAS-4000 (Fuji film, Tokyo, Japan) and analyzed using ImageJ software (NIH, Bethesda, MD, USA).
3
Immunohistochemical Analysis of Fibronectin in Cuprizone-Treated Mice
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Paraffin-embedded brains of cuprizone-treated TG2−/− mice and littermate controls were cut in 5 μm sections at the level of the corpus callosum. Sections were deparaffinised in xylene and transferred to distilled water via degrading 90%, 70% and 50% ethanol series. As a pre-treatment, sections were incubated with 20 μg/ml proteinase K (Sigma P2308) in TBS containing 0.02 M CaCl2 for 15 min at 37 °C. All further incubation steps were performed in DAKO washing buffer (Dako, Denmark) with additional 10% fetal calf serum. The sections were incubated overnight at 4 °C with a goat-anti-human fibronectin antibody (1:50; Santa Cruz, C-20). Subsequently, sections were incubated for 1 hr at room temperature with biotinylated donkey anti-goat IgG’s (1:200; Jackson ImmunoResearch Laboratories Inc). Sections were washed in TBS and incubated with horse radish peroxidase (HRP)-labelled avidin (1:100; Sigma) for 1 hr at RT. Finally, peroxidase activity was visualized using 3,3-diaminobenzidine (DAB, Sigma, St. Louis, USA) as a chromogen.
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