K3468

Manufactured by Agilent Technologies

Sourced in Denmark, United States, Belgium

The K3468 is a laboratory equipment product manufactured by Agilent Technologies. It is designed to perform specific functions within a laboratory setting. The core function of the K3468 is to provide precise and reliable measurements for research and analytical applications.

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Lab products found in correlation

104 protocols using k3468

Immunohistochemical Staining of Esophageal Cancer

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Thin slices were made from a paraffin block of esophageal cancer tissue and mounted on a glass slide. After deparaffinization, sections were incubated with antigen activator (Dako S2031, Tokyo, JAPAN) with 10-fold dilution for 40 min at 100°C. Sections then were left at room temperature for 20 min. After washing with water, sections were kept in distilled water for 5 min. The sections, topped with 3% hydrogen peroxide containing methanol (Dako S2023, Tokyo, JAPAN), were kept at room temperature for 30 min followed by washing with wash buffer (Dako S3006, Tokyo, JAPAN) three times at 5 min per washing, followed by incubation with primary antibody diluted with dilution buffer (Dako S0809, Tokyo, JAPAN) for 60 min at 37°C or overnight at 4°C. After incubation with primary antibody, sections were washed three times at 5 min per washing with the Wash Buffer (Dako S0809, Tokyo, JAPAN). Secondary antibody (ENVISION+/HRP-labeled Dako K5007, Tokyo, JAPAN) was added and sections were incubated for 60 min at 37°C. After incubation with diaminobenzidine (DAB; Dako K3468, Tokyo, JAPAN) for color development, sections were washed and kept in distilled water for 5 min. After nuclear staining with hematoxylin for 1 min, sections were washed with running water followed by dehydration, cleared, and sealed with a cover glass. The sections were examined under an optical microscope.

Ogura Y., Hoshino T., Tanaka N., Ailiken G., Kobayashi S., Kitamura K., Rahmutulla B., Kano M., Murakami K., Akutsu Y., Nomura F., Itoga S., Matsubara H, & Matsushita K. (2018). Disturbed alternative splicing of FIR (PUF60) directed cyclin E overexpression in esophageal cancers. Oncotarget, 9(33), 22929-22944.

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Immunohistochemical Analysis of Breast Tissue

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Assembly and immunohistochemistry staining of human breast tissue microarrays (TMAs) were performed by the Biorepository and Tissue Analysis core at MUSC. TMAs consisted of matched normal and tumor tissue as well as normal and metastatic lymph node biopsies from patients for whom survival data were available (Supplementary Table S3). For immunohistochemistry staining of formalin-fixed, paraffin-embedded TMAs, sections were deparaffinized in xylene, rehydrated in alcohol and processed as follows: sections were incubated with target retrieval solution (S2367; Dako) in a steamer for 45 min followed by 3% hydrogen peroxide solution for 10 min and protein block (X0909; Dako) for 20 min at room temperature. Sections were incubated overnight in a humidity chamber at 4°C with α-Cdc27 antibody [AF3.1] (ab10538; Abcam) or α-Fzr1 (ab3242; Abcam) followed by biotinylated secondary antibody (PK6106; Vector laboratories) for 30 min and ABC reagent for 30 min. Immunocomplexes of horseradish peroxidase were visualized by DAB reaction (K3468; Dako), and sections were counterstained with hematoxylin before mounting. Images of stained sections were taken using an Olympus DP72 8-bit RGB camera with Cellsens acquisition software.

Link L.A., Howley B.V., Hussey G.S, & Howe P.H. (2016). hnRNP E1 Protects Chromosomal Integrity by Translational Regulation of Cdc27. Molecular cancer research : MCR, 14(7), 634-646.

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Immunohistochemistry for Melan A and H4K20me3

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This was performed as described previously [81 ]. Briefly, formalin-fixed, paraffin-embedded sections were deparaffinized, rehydrated, and blocked for endogenous peroxidases and underwent antigen retrieval according to antibody specifications. Tissues were incubated overnight with the following primary antibodies: anti-human melan A clone A103 (M7196; Dako), anti-H4K20me3 (04–079, Millipore and cs5737, Cell Signaling). Secondary antibodies used for 3,3′-diaminobenzidine (DAB)-based immunohistochemistry were either EnVision + System-HRP Labeled Polymer Anti-mouse (K4001; Dako) or EnVision + System-HRP Labeled Polymer Anti-rabbit (K4003; Dako) based on primary antibody host species. Peroxidase activity was revealed using DAB (K3468; Dako). Samples were then counterstained with hematoxylin, dehydrated, and coverslipped.

Nelson D.M., Jaber-Hijazi F., Cole J.J., Robertson N.A., Pawlikowski J.S., Norris K.T., Criscione S.W., Pchelintsev N.A., Piscitello D., Stong N., Rai T.S., McBryan T., Otte G.L., Nixon C., Clark W., Riethman H., Wu H., Schotta G., Garcia B.A., Neretti N., Baird D.M., Berger S.L, & Adams P.D. (2016). Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability. Genome Biology, 17, 158.

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Immunohistochemical Analysis of IL4I1 and SP1

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The sections of formalin-fixed and paraffin-embedded tumor tissues were dewaxed and subjected to heat-mediate antigen retrieval using EDTA Antigen Retrieval Solution (Beyotime, China). After incubation of 3% H₂O₂ to inactivate endogenous peroxidase for 15 min and 10% normal goat serum to block nonspecific sites for 30 min, sections were incubated with antibodies against IL4I1 (1:100, ab222102, Abcam), SP1 (1:100, 9389S, CST) at 4°C overnight. Sections were then stained with K5007Dako REAL EnVision detection kit for 45 min and visualized using 3,3′-diaminobenzidine (DAB, K3468, DAKO, Denmark). The images were obtained by KF-Pro-020 pathological section scanner (KFBIO, China).

Zhang G.P., Xie Z.L., Jiang J., Zhao Y.T., Lei K., Lin Z.L., Chen S.L., Su T.H., Tan L., Peng S., Wang J., Liu C, & Kuang M. (2023). Mechanical confinement promotes heat resistance of hepatocellular carcinoma via SP1/IL4I1/AHR axis. Cell Reports Medicine, 4(8), 101128.

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Immunohistochemical Analysis of SGLT1 and Phospho-SGK1 in Tamoxifen-Treated Mouse Intestine

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Formalin (10%)-fixed paraffin-embedded jejunal tissues from tamoxifen (2 mg/day/mouse)-treated C57BL/6J (WT), R26^CreER;MYO5B^f/wt (Het), and R26^CreER;MYO5B^f/f (Homo) mice were sectioned at 5~8 μm of thickness and sections were deparaffinized in xylene and rehydrated through ethanol to distilled water. Antigen retrieval was performed with citrate buffer (DAKO, Carpinteria, CA, USA) for 30 min. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 5 min at room temperature. The slides were rinsed with Tween-20-Tris-buffered saline solution between each of the following steps. The sections were incubated with primary antibodies (SGLT1 and Phospho-SGK1^Thr256) overnight at 4 °C. The antibodies were then detected by use of EnVision+ (#K4001, DAKO) solution. Diaminobenzidine was used to detect the antibody complex (#K3468, DAKO). Negative control sections were incubated with isotype-matched immunoglobulins. The slides were subsequently counterstained with hematoxylin, dehydrated, and cover slipped with resin mounting media. Immunolabeled sections were examined by light microscopy on an Olympus BX51 at ×40 magnification. Digital images were acquired with an Olympus cell Sens Standard imaging software.

Ahsan M.K., dos Reis D.C., Barbieri A., Sumigray K.D., Nottoli T., Salas P.J, & Ameen N.A. (2022). Loss of Serum Glucocorticoid-Inducible Kinase 1 SGK1 Worsens Malabsorption and Diarrhea in Microvillus Inclusion Disease (MVID). Journal of Clinical Medicine, 11(14), 4179.

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Immunohistochemical Detection of MVH

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Immunostaining was performed using a Lab Vision Autostainer 360 (Thermo Scientific) as per manufacturer's instructions. Briefly, a citrate-based solution (S1699; Dako) was used for epitope retrieval at ∼95°C in a steamer for 20 minutes. A 3% H2O2/PBS solution was used for endogenous enzyme block. Once in the autostainer, slides were incubated with 2.5% normal horse serum for 20 minutes then incubated with rabbit anti-MVH (Ab13840; Abcam) diluted to 1 µg/mL in Dako Antibody Diluent (S3022) for 30 minutes, rinsed, and then incubated with ImmPRESS Horse Anti-Rabbit HRP (MP-7401; Vector Laboratories) for 30 minutes. DAB (K3468; Dako) was applied for 2.5 minutes.

Cuitiño M.C., Fleming J.L., Jain S., Cetnar A., Ayan A.S., Woollard J., Manring H., Meng W., McElroy J.P., Blakaj D.M., Gupta N, & Chakravarti A. (2023). Comparison of Gonadal Toxicity of Single-Fraction Ultra-High Dose Rate and Conventional Radiation in Mice. Advances in Radiation Oncology, 8(4), 101201.

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HCV Core 1b Protein Detection Assay

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J6/JFH1-huh 7.5 cells were plated with a density of 5 × 10⁴ cells/100 μL cells per well in 96-well plate and fixed overnight before being permeabilized in 100% methanol for 30 min at −20 °C. Cells were washed twice with PBS followed by once with 1× PBS/0.1% Tween-20 and blocked with 1% BSA and 0.2% skim milk for 30 min at room temperature. Then, 3% H₂O₂ was added to block endogenous peroxidase activity. Cells were stained with anti-HCV-specific Core 1b monoclonal antibody (#ab2740, Abcam, Cambridge, UK) diluted 1:2000 in 1× PBS/0.1% Tween-20 and incubated with secondary antibody (goat-a-mouse-HRP, Jackson Immuno Research) diluted 1:200 for 30 min at room temperature. DAB substrate (DAKO, K3468; diluted 1 drop/mL as per manufacturer’s instructions) was added for 5 min to detect positive dark spots. Also, nuclei counterstain was done using Gill’s Hematoxylin #2 (Polysciences, Inc., Warrington, PA, USA) to confirm the absolute cell number.

Cho Y.J., Lee H.H., Kang H, & Cho H. (2018). Cytokine-Modulated Natural Killer Cells Differentially Regulate the Activity of the Hepatitis C Virus. International Journal of Molecular Sciences, 19(9), 2771.

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Immunohistochemical Evaluation of Cell Markers

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Paraffin-embedded tissues (5 μm thick) were subjected to immunohistochemical staining as previously reported [46 (link)]. Anti-phosphorylated histone H3 (PH-H3, 06-570, Millipore), anti-survivin (2808, Cell Signal Technologies), and anti-SIRT1 (sc-74504, Santa Cruz Biotechnology) were incubated at 4 °C overnight. Secondary antibody development was performed with Dako Cytomation EnVision + mouse or rabbit labeled polymer kits (K4001 and K4003, Dako Cytomation) and visualized using 3,3′-diaminobenzidine tetrahydrochloride substrate (K3468, Dako Cytomation). Up to five fields per tissue (n = 4) were quantitated with Fiji (ImageJ) using a custom-written macro. Mitotic (PH-H3) index was calculated by taking the total number of positive (brown) nuclei divided by the total number of nuclei.

Madhav A., Andres A., Duong F., Mishra R., Haldar S., Liu Z., Angara B., Gottlieb R., Zumsteg Z.S, & Bhowmick N.A. (2018). Antagonizing CD105 enhances radiation sensitivity in prostate cancer. Oncogene, 37(32), 4385-4397.

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Immunohistochemical Analysis of Hepatic α-SMA

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Five-µm-thick hepatic tissue sections were deparaffinized in xylene and rehydrated serially with alcohol and water, followed by microwave antigen retrieval for 20 min at 98 °C in 10 mM sodium citrate buffer (0.05% tween 20, pH 6.0). Endogenous peroxidase was blocked with fresh 0.3% hydrogen peroxide in phosphate buffered saline (PBS) for ten minutes at room temperature. Sections were blocked 2 h with protein block (DAKO, Belgium) in PBS containing 0.5% triton and were incubated overnight at 4 °C in the presence of a specific alpha-smooth muscle actin antibody (α-SMA, 1/200, ab5694, Abcam, UK). After being washed three times in PBS, sections were incubated with biotinylated secondary swine anti-rabbit antibody (E0431, DAKO, Belgium) for 1 h and with streptavidine-HRP (1/800, P0397, DAKO, Belgium) for 30 min. α-SMA was visualized using DAB (K3468, DAKO, Belgium) and sections were counterstained with hematoxylin. Sections were observed at 20X magnification Mirax Desk and Mirax viewer, Carl Zeiss MicroImaging, Germany).

Cops J., Mullens W., Verbrugge F.H., Swennen Q., De Moor B., Reynders C., Penders J., Achten R., Driessen A., Dendooven A., Rigo J.M, & Hansen D. (2018). Selective abdominal venous congestion induces adverse renal and hepatic morphological and functional alterations despite a preserved cardiac function. Scientific Reports, 8, 17757.

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Quantitative Immunohistochemical Analysis

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Paraffin embedded tissues (5 µm thick) were subjected to immunohistochemical staining as previously reported (46 (link)). Anti-phosphorylated histone H3 (PH-H3, 06-570, Millipore), anti-survivin (2808, Cell Signal Technologies), anti-SIRT1 (sc-74504, Santa Cruz Biotechnology) were incubated at 4°C overnight. Secondary antibody development was performed with Dako Cytomation EnVision+ mouse or rabbit labeled polymer kits (K4001 and K4003, Dako Cytomation) and visualized using 3,3’-diaminobenzidine tetrahydrochloride substrate (K3468, Dako Cytomation). Up to five fields per tissue (n=4) were quantitated with Fiji (ImageJ) using a custom written macro. Mitotic (PH-H3) index was calculated by taking the total number of positive (brown) nuclei divided by the total number of nuclei.

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