RAGE immunohistochemistry was performed on paraffin liver sections (7 µm) of db/db and db/+ mice (n=3) using the Dako EnVision™+ Dual Link System-HRP kit according to the manufacturer's instructions (K4065; DAKO, Glostrup, Denmark A⁄ S, Denmark) with minor modifications. Briefly, sections were deparaffinized, rehydrated and an antigen retrieval was performed in sodium citrate buffer (pH 6; 0.01 M) for 2 min in a microwave (700 W). Sections were incubated overnight at room temperature with the RAGE antibody (1/ 100; Abbiotec; Ref: 251890) in PBS-Triton (0.05%) containing 5% BSA. The next day, after incubation with the labeled polymer HRP (K4065; DAKO) and staining with the substrate chromogen 3,3′diaminobenzidine tetrahydrochloride (DAB) (K4065; DAKO), sections were counterstained with DAPI and slides mounted with the antifading medium Vectashield (Vector Laboratories, Burlingame, CA).
K4065
Manufactured by Agilent Technologies
Sourced in Denmark, United States
The K4065 is a laboratory equipment product from Agilent Technologies. It is a high-precision device designed for specific laboratory applications. The core function of the K4065 is to perform measurement and analysis tasks within the intended scope of use. No further details about the product's specific capabilities or intended applications are provided.
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Lab products found in correlation
E600 microscope, by Nikon (1 mentions)
Opal system, by Akoya Biosciences (1 mentions)
Aquatex, by Merck Group (1 mentions)
Ab109131, by Abcam (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Ab4059, by Abcam (1 mentions)
Ab33786, by Abcam (1 mentions)
Ab23990, by Abcam (1 mentions)
Anti tgf β, by Abcam (1 mentions)
α sma, by Agilent Technologies (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti il 6, by Abcam (1 mentions)
Anti tnf α, by Abcam (1 mentions)
Envision dual link system hrp kit, by Agilent Technologies (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
M7248, by Agilent Technologies (1 mentions)
K5007, by Agilent Technologies (1 mentions)
7 protocols using k4065
1
RAGE Immunohistochemistry in Diabetic Liver
2
Histological Examination of Pancreatic Tissue
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For the histological examination, pancreatic tissues were fixed in 4% paraformaldehyde, and 3 μm paraffin sections were stained with haematoxylin and eosin (H&E). To determine islet insulin content, paraffin sections were stained with primary antibodies (1:200) against insulin (Merck Millipore, KGaA, Darmstadt, Germany). After washing, a secondary antibody conjugated to horseradish peroxidase (Dako EnVision K4065, Glostrup, Denmark) was used. The result was visualized using the DAB reagent (Dako EnVision) in the presence of Mayer’s haematoxylin staining. Images were captured using a Nikon E600 microscope that is equipped with a digital camera (SAGE vision SG-5.07, Nikon, Tokyo, Japan) [41 (link)].
3
Multiplex Immunofluorescence Staining Protocol
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We used the OPAL™ system (Akoya Biosciences) for staining the following target combinations: TSPO/CD68, TSPO/CD11b, TSPO/GFAP, and p53/TSPO. Briefly, paraffin sections (3 μm) were baked (50 °C), deparaffinized, and rehydrated. First target stain: HIER was performed for 30 min. After washing (PBS, 0.05% Tween® 20), sections were blocked for 10 min with kit-provided endogenous enzyme block (K4065, Dako) at room temperature (RT). Unspecific binding sites were blocked with 1% bovine serum albumin (10 min, RT). Incubation with primary antibodies was performed (1 h, RT). After washing, sections were labeled with Opal polymer HRP Ms + Rb (10 min, RT). After additional washing, sections were incubated with Opal Dye working solution (10 min, RT). Specifications for HIER buffers, primary antibodies and OPAL fluorescence dyes/work solution are provided in Suppl. Table 5. After washing again, the second target stain was performed similarly to the first one. Then, a final HIER step in 10 mM citrate buffer (pH 6.0) was followed by nuclear counterstaining using Spectral DAPI solution (1 droplet/ml TBST, pH 7.5, 0.05% Tween® 20) for 5 min at room temperature. Finally, slides were cover-slipped using Aquatex® (#1.08562.0050, Merck KGaA) and JPEG images (400x) were counted within Fiji [64 (link)] with the Cell Counter plugin [39 ].
4
Immunohistochemical detection of ACE2 and TMPRSS2
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All paraffin wax blocks were cut at 4 μm and the neighboring sections were used. Slides were dewaxed and re-hydrated with xylene and different concentration of ethanol. Slides were immersed in sodium citrate buffer (pH 6.0) and boiled for 10 min in a microwave oven. After treatment with 3% hydrogen peroxide for 10 min, sections were blocked with 1% milk and reacted with first antibodies against ACE2 (tcna2043, 1:800 dilution; Taiclone, Taiwan) and TMPRSS2 (ab109131, 1:500 dilution; Abcam, USA) for overnight at 4 °C. After washes, sections were subsequently incubated with biotinylated secondary antibodies (K4065; Dako, USA) for 1 h at room temperature. Slides were stained using 3,3′-diaminobenzidine chromogen (DAB) solution and counterstained with haematoxylin, followed by mounting. We photographed all of the lung tissues in the slides by microscopy (Nikon, USA). About 4–6 measurements of 40X magnification for each subject sample (n = 4 per group) were analyzed by image-analysis system (Image Pro-Plus, Media Cybernetics, USA). The labeled images were based on RGB 8-bit resolution per channel parameters. The segmented areas in the images were filtered to count stained area. Mean density and area thresholds were automatically defined based in the assessed images. After application, the area labeled and their counting per image was obtained [21 ].
5
Quantifying Immune Cells in Tumor Samples
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Immunohistochemistry (IHC) was used in the formalin-fixed and paraffin-embedded tumor sections to determine the numbers of CD8A, GZMB, MHC1 and MHCII positive cells, by using a secondary antibody and visualization system (K4065, DAKO). Antibodies used and dilutions in PBS were as follows: CD8A (1:100, ab33786, Abcam), GZMB (1:100, ab4059, Abcam), MHCI (1:100, NB120-6405, Novus Biological) and MHCII (1:50, ab23990, Abcam). Tissue staining was examined under a bright field microscope and 20–30 pictures for CD8A and GZMB were captured from each sample to represent the entire section. For MHCI and MHCII, 16–256 pictures were captured depending on the tumor size. Positive stained cells were quantified by a macro function in ImageJ and the average CD8A+ , GZMB+, MHCI+ and MHCII+ cells were calculated. Staining was considered positive when >10% of total cells were positive.
6
Tissue Microarray Immunohistochemistry Protocol
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The area of ulcer/scar was marked on a slide for the tissue microarray procedure. The slide was paired with a para n block and a tissue microarrayer (Quick-Ray Unitma Co. Ltd., Seoul, Korea) was used to punch a sample (2-mm diameter) from the para n block. The sample was transferred to receptor para n blocks with capacity for 36 samples each [adapted from 24]. Sections (2.5 µm thick) were immunoreacted with the primary antibodies anti-TNF-α (1:100), anti-IL-6 (1:300), anti-IL-1β (1:150) and anti-TGF-β (1:400) (Abcam ™ ) and α-SMA (1:400) (Dako ™ ). Slides were incubated with biotinylated readyto-use, monoclonal anti-rabbit IgG secondary antibody, at room temperature for 30 min (K4065, Dako ™ ). Paired sections were treated with the control IgG in substitution to the primary antibody. Staining intensity was measured according to the following scores: (0) no cells; (1) mild, 1-33% cells; (2) moderate, 34-66% cells; (3) 67-100% cells [adapted from 22] .
7
Multicolor Immunohistochemistry for Glioma
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Vibratome sections (200 μm thick) of mouse brains were paraffin-embedded and then consecutive semithin sections were processed and incubated with primary antibodies against Vimentin, glial fibrillary acidic protein (GFAP) and Ki67 (M7248, DAKO) followed by horseradish peroxidase-conjugated secondary antibodies (K4065, K5007, DAKO) and visualized by a chromogen or stained with hematoxilin-eosin. Slides containing tumor tissue from GBM patients or neurospheres from GSC cultures embedded in paraffin were stained with antibodies against the N-terminal region of ODZ1.
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