Ectopic endometriotic lesion (n = 3) and eutopic endometrium (n = 3) were embedded in paraffin and cut (5 µm) and mounted on charged slides (Superfrost, Thermoscientific, Canada). Paraffin-embedded endometrium were used for TTP and HuR immunolocalization experiments using previously published IHC protocol25 . Briefly, antigen retrieval was performed with citric acid buffer boiling for 2 mins. Blocking was performed on all sections using 1% bovine serum albumin (BSA) for 1.5 hours at room temperature. Rabbit polyclonal to tristetraprolin (sc-12563, Santa Cruz Biotechnology, USA) and Rabbit monoclonal anti-HuR (ab200342, Abcam plc., UK) were added at 1.5 µg/mL and 0.8 µg/mL, respectively. Biotin-conjugated secondary antibody was used (K0690, DakoCytomation, USA) was added to all sections and incubated for 45 mins at room temperature. DAB was utilized as chromogen and was controlled for 10 seconds and 15 seconds per section for TTP and HuR, respectively. Counterstaining was done using haematoxylin for 15 seconds and upon rinsing, slides were dehydrated and coverslips were added. All slide images were taken using a ZEISS observer microscope and analyzed using ZEISS imaging software (Zeiss, Canada).
K0690
Manufactured by Agilent Technologies
Sourced in United States
The K0690 is a laboratory instrument designed for conducting spectroscopic analysis. It is capable of measuring the absorption, emission, or reflection of light by samples across a range of wavelengths. The core function of the K0690 is to provide accurate and reliable data for various analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab200342, by Abcam (1 mentions)
K3465, by Agilent Technologies (1 mentions)
S2020, by Agilent Technologies (1 mentions)
Mayer s hematoxylin, by Agilent Technologies (1 mentions)
Il 1β, by Santa Cruz Biotechnology (1 mentions)
Tnf α, by Santa Cruz Biotechnology (1 mentions)
Lsab system hrp, by Agilent Technologies (1 mentions)
Il 17, by Santa Cruz Biotechnology (1 mentions)
Liquid dab, by Agilent Technologies (1 mentions)
K3468, by Agilent Technologies (1 mentions)
M7249, by Agilent Technologies (1 mentions)
5 protocols using k0690
1
Immunolocalization of TTP and HuR in Endometriosis
2
Immunolocalization of NeuGcGM3 Ganglioside
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Five-micrometer serial sections from each block were obtained in a micrometer (Leitz, 1512) and they were mounted on plus slides (Dako, S2024). All sections were attached to the slide by heating in a 60°C oven for 1 h. Afterward, the slides were dewaxed in xylene and rehydrated in graded ethanol series in the usual way. The samples were maintained in tap water until they were stained.
The immunolocalization of NeuGcGM3 ganglioside was performed as it was previously described in [10 (link)] with some modifications. Briefly, the slides were incubated with 14F7 Mab in a humid chamber for 1 h at room temperature followed by the labeled streptavidin biotin (LSAB) two steps' system (Dako, K0690) both for 30 minutes at room temperature. The enzymatic activity was visualized with 3,3-diaminobenzidine (DAB) substrate chromogenic solution (Dako, K3465) and the tissues were counterstained with Mayer's Hematoxylin (Dako, S2020). Concerning the evaluation of both EGFR and EGF tissue antigens, the procedure as it was previously described in [19 (link)] was used.
The immunolocalization of NeuGcGM3 ganglioside was performed as it was previously described in [10 (link)] with some modifications. Briefly, the slides were incubated with 14F7 Mab in a humid chamber for 1 h at room temperature followed by the labeled streptavidin biotin (LSAB) two steps' system (Dako, K0690) both for 30 minutes at room temperature. The enzymatic activity was visualized with 3,3-diaminobenzidine (DAB) substrate chromogenic solution (Dako, K3465) and the tissues were counterstained with Mayer's Hematoxylin (Dako, S2020). Concerning the evaluation of both EGFR and EGF tissue antigens, the procedure as it was previously described in [19 (link)] was used.
3
Immunohistochemical Analysis of Cytokines
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Histological sections were mounted on silanized slides to perform the reactions according to a previously described protocol [8 (link), 19 (link)]. The following primary antibodies were used: IL-1β (1 : 100, rabbit, sc-7884), IL-17 (1 : 200, rabbit, sc-7927), and TNF-α (1 : 50, mouse, sc-130349) from Santa Cruz Biotechnology (Dallas, TX). The secondary antibody LSAB + HRP system (Dako K0690, DAKO North America, and Carpinteria, CA) was used. To stain the areas of cytokine expression, Liquid DAB (Dako K3468, DAKO North America, and Carpinteria, CA) was used. Following the manufacturers' instructions, positive and negative controls were used for each antibody.
4
Immunohistochemical Staining of Mouse Organs
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Formalin-fixed, paraffin embedded sections of mouse organs were stained with primary antibody CIP2A [31 (link)], ki-67 (M7249, Dako) or peanut agglutinin (PNA, L6135, Sigma-Aldrich). Secondary antibody (Dako EnVision anti-rat) or HRP conjugated streptavidin from the LSAB+ skit (K0690, Dako) were used for visualisation. DAB+ liquid Dako (K3468) was used as a substrate for peroxidase. Samples were incubated in Mayers HTX, rinsed and finally dehydrated, cleared and mounted.
5
PRAME Immunohistochemistry in Breast Tumors
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For the IHC; 0.5 mm thick tissue sections from paraffin-embedded breast tumor samples were used. After deparaffinization, the sections were heated in a 600 w household microwave oven for 50 minutes in EDTA buffer (PH: 8) and washed with phosphatebuffered saline (PBS; PH 7.2). After an additional PBS wash, the sections were incubated for 20 minutes with 1:10 diluted normal rabbit sera (DAKOX902) at room temperature in a humidified chamber to prevent non-specific immunoglobulin binding. The sections were treated with primary antibody (Rabbit polyclonal Ab to PRAME cat no: 32185) for 3 hours at room temperature. A streptavidin-biotinylated horseradish peroxidasebased detection system (DAKO K 0690) was used to reveal specific binding. Testicular tissue with intact spermatogenesis was used as positive control. Nonneoplastic ductal epithelial cells were indeed present in all specimens, as internal controls. The staining of invasive, in-situ component, and normal breast tissue were evaluated separately. IHC staining of PRAME was visible as cytoplasmic/nuclear staining limited to tumor cells. Immunoreactivity of tumor cells was graded as follows: 0 (no positive tumor cells), + (<25% positive tumor cells), ++ (25-50% positive tumor cells), and +++ (>50% positive tumor cells).
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