Tissues were fixed using 4% formalin for 48 h. Then, the tissues were embedded using OCT (Sigma, USA). The tissues were sectioned in 8 μm thickness. After antigen repair, 3% H2O2 treatment, washing with PBS, and blocking with 5% goat serum. The primary antibodies (Rabbit monoclonal to c-myc, 1:1500, #ab32072, Abcam; Rabbit monoclonal to axin, 1:1000, #ab109307, Abcam; Rabbit monoclonal to beta catenin, 1:1000, #ab32572, Abcam) were used to cultivate tissues at 4 °C overnight. After washing, the tissues were cultured with second antibody (Goat anti-rabbit lgG, 1:2000, #ab205718, Abcam) for 2 h. DAB reagent (#34002, Thermo Fisher, USA) was used to treat tissues. The sections were visualized and captured using an inverted optical microscope (Olympus Corporation). The expression intensity was analyzed through Image J software.
Dab reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The DAB reagent is a laboratory product used for immunohistochemistry and in situ hybridization applications. It provides a brown chromogenic reaction that visually indicates the presence of a target analyte in a biological sample.
Automatically generated - may contain errors
Lab products found in correlation
Ab109307, by Abcam (1 mentions)
Inverted optical microscope, by Olympus (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab32072, by Abcam (1 mentions)
Coomassie brilliant blue, by Beyotime (1 mentions)
Anti cchfv pre gc mab clone 11e7, by BEI Resources (1 mentions)
Nitrocellulose membrane, by Beyotime (1 mentions)
Glt 1, by Abcam (1 mentions)
Glast, by Cell Signaling Technology (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
5 protocols using dab reagent
1
Immunohistochemical Analysis of Cell Signaling
2
Brain Immunohistochemistry for PrP Detection
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Four-µm sections of brain were cut onto positively charged silanized glass slides and stained with hematoxylin and eosin or immunostained using antibodies for PrP (12F10). For PrP staining, sections were deparaffinized and incubated for 5 min in 96% formic acid, then washed in water for 5 min, treated with 5 µg/ml of proteinase K for 7 min, and washed in water for 5 min. Sections were then placed in citrate buffer (pH 6) and heated in a pressure cooker for 20 min, cooled for 5 min, and rinsed in distilled water. Sections were incubated with anti-PrP 12F10 (Cayman Chemical; 1:200) for 45 min followed by anti-mouse IgG conjugated to biotin (Jackson Immunolabs; 1:250) for 30 min, followed by streptavidin-HRP (Jackson Immunolabs; 1:2,000) for 30 min. Sections were then incubated with DAB reagent (Thermo Fisher Scientific) and counterstained with hematoxylin.
3
Determining Molecular Weight of rGP
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The molecular weight of the truncated rGP was determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting and described as follows. Two BeyoGel™ Plus PAGE 12% tris-acetate protein gels (Beyotime, Shanghai, China) were run at a constant 130 V for 90 min: one was stained with Coomassie brilliant blue (Beyotime, Shanghai, China), and the other was transferred onto the nitrocellulose membrane (Beyotime, Shanghai, China) according to the manufacturer’s instructions. After the immobilization and blocking of the bands, the membrane was incubated overnight at 4°C with the anti-CCHFV pre-GC mAb clone 11E7 (BEI Resources, Manassas, VA, USA). After washing three times with PBST, the membrane was treated with horseradish peroxidase (HRP)-conjugated anti-mouse-specific IgG antibody (Bioworld, Minnesota, MN, USA; diluted 1:5,000) at room temperature for 1 h. After another three washes with PBST, the bands were visualized after incubation with DAB reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions.
4
Immunohistochemical Analysis of Hippocampal Astrocytes
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Immunohistochemistry staining was carried out according to the method previously described [11 (link)]. The brains were fixed in 4% PFA solution for 48 h and were then cut into serial sections (5 μm). The paraffin-embedded hippocampal sections were processed as free-floating slices, including deparaffinized, rehydrated, and pretreated with hydrogen peroxidase. Antigen retrieval was performed by heating in 0.01 mmol/L citrate buffer (PH = 7.2) for 15 min. Slices were incubated with primary antibodies (GLAST, Cell signaling, #5684, 1:50; GLT-1, Abcam, #69098, 1:300) after blocking in the antisera. After incubation with the secondary antibody, sections were placed in DAB reagent (#00–2014, Invitrogen, USA) for 5–10 min at room temperature. After a further rinsing in 0.1 mol PBS, sections were restained with hematoxylin, and were mounted on gelatine-coated slides for observation. The images of the positively stained expressions in the CA1, CA3 and DG regions of the hippocampus were captured at 200 x magnifications by an Olympus BX 41 microscope.
5
Immunohistochemical Analysis of Brain Tissue
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Four μm sections of brain were cut onto positively charged silanized glass slides and stained with hematoxylin and eosin, or immunostained using antibodies for PrP (SAF84) or GFAP for astrocytes. For PrP staining, sections were deparaffinized and incubated for 5 min in 96% formic acid, then washed in water for 5 min, treated with 5 μg/ml of proteinase-K for 7 min, and washed in water for 5 min. Sections were then placed in citrate buffer (pH 6) and heated in a pressure cooker for 20 min, cooled for 5 min, and washed in distilled water. Sections were blocked and incubated with anti-PrP SAF-84 (SPI bio; 1:400) for 45 min followed by anti-mouse biotin (Jackson Immunolabs; 1:250) for 30 min, followed by streptavidin-HRP (Jackson Immunolabs; 1:2000) for 30 min. Slides were then incubated with DAB reagent for 7 min and an enhancer for 2 min (Invitrogen). Sections were counterstained with hematoxylin. GFAP immunohistochemistry for astrocytes (1:500; DAKO) was similarly performed, however with antigen retrieval by PK-digestion (20 μg/ml for 10 min at room temperature).
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