Paraffin-embedded patient tissue blocks sectioned at 4 µm thickness. The slides were allowed to dry for a day and were left to warm at 60 °C degrees for an hour. For antigen retrieval, 3% H2O2 and 95 °C antigen retriever buffer were used. For permeability, 0.2% triton solution was treated and for blocking, 5% BSA in PBS was treated for 15 min. The 1st antibody was treated with Rabbit Polyclonal antibody SOCS4 (1:100, Mybiosource, MBS7043907) and incubated at 4 °C overnight. Additionally, Goat anti-Rabbit IgG (H + L), HRP (1:100, Thermofisher, A11008) was treated as a secondary antibody for 1 h at room temperature. Stained with DAB Substrate Kit (3,3′-diaminobenzidine, VECTORLABS, SK-4100) and counterstaining with 50% Hematoxylin for 30 s. Then, the slide was dried at 37 °C for 1 h (Mounted with Eukitt® Quick-hardening mounting medium (Sigma Aldrich, St. Louis, MO, USA, 03989-100)). Slides were interpreted twice by two pathologists.
Eukitt quick hardening mounting medium
Manufactured by Merck Group
Sourced in United States, Germany
Eukitt Quick-hardening mounting medium is a laboratory product designed to provide a quick-hardening solution for mounting samples on microscope slides. It is a clear, resinous medium that helps preserve and protect specimens for microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost plus slide, by Thermo Fisher Scientific (4 mentions)
Nis elements ar software, by Nikon (3 mentions)
Tie 3000 inverted microscope, by Nikon (3 mentions)
Ds u2 l2 ri1 digital microscope camera, by Nikon (3 mentions)
Dxm 1200c coded digital camera, by Nikon (3 mentions)
Lumar v12 stereomicroscope, by Zeiss (3 mentions)
Mayer s hematoxylin, by Agilent Technologies (2 mentions)
Axioskop microscope, by Zeiss (2 mentions)
Axiocam camera, by Zeiss (2 mentions)
Nuclear fast red, by Avantor (2 mentions)
X gal, by Promega (2 mentions)
Picric acid solution 1 3, by Merck Group (2 mentions)
5000b microscope, by Leica (2 mentions)
Direct red 80, by Merck Group (2 mentions)
Db5000, by Leica (2 mentions)
Bx61 upright microscope, by Olympus (2 mentions)
Fd rapid golgistain kit, by FD NeuroTechnologies (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg h l hrp, by Thermo Fisher Scientific (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
56 protocols using eukitt quick hardening mounting medium
1
Immunohistochemical Analysis of SOCS4 in Paraffin-Embedded Tissues
2
Quantifying Hepatic Collagen Deposition
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To assess the degree of fibrosis, tissue sections were stained for collagen 1 and 3 fibers using Picro-Sirius red (Abcam). Briefly, thick liver sections (4–6 μm) were deparaffinized and incubated for 1 h with Picro-Sirius red. The sections were rinsed twice in acetic acid and dehydrated by dipping twice in absolute alcohol. Sections were mounted with Eukitt quick hardening mounting medium (Sigma-Aldrich) and visualized using a Zeiss 510 microscope (Carl Zeiss, Thornwood, NY, USA) equipped with a 20X objective. Five random fields from each animal group were visualized and relative staining of collagen was assessed in a blinded fashion. The percent of fibrosis was calculated based on the intensity of Sirius red staining using ImageJ (NIH, Bethesda, MD, USA).
3
Metaphase Chromosome Spreading Assay
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SCEs were analysed as described previously (36 (link)). Briefly, cells were grown for 48 h with the addition of 10 μM BrdU. To increase the number of mitotic cells, 1 μM nocodazole was added for the last 16 h before the cells were collected. Cells were swollen in 75 mM KCl at 37°C before being fixed with 3:1 methanol: acetic acid and spotted onto glass slides. After being allowed to dry cells were stained with 10 μg/ml Hoechst. Slides were washed in SSC, (150 mM sodium chloride, 15 mM sodium Citrate) and exposed to UV light for 1 h before being incubated in SSC buffer for a further 1 h at 60°C. Finally, slides were stained with Giemsa and after drying, mounted with Eukitt Quick-hardening mounting medium (Sigma-Aldrich). Slides were then imaged and counted as described above.
4
Quantification of Malaria Parasitemia
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Thick blood films from samples positive for malaria infection by nested PCR assays were stained with 3% Giemsa for 45 min, air dried, mounted using Eukitt® Quick-hardening mounting medium (Sigma-Aldrich, Germany) and examined by microscopy to observe for parasites and the parasite life cycle stage. The number of parasites in 1 μl of blood was calculated based on the formula below [33 ]:
Photos of malaria parasites observed were captured using a 500 megapixel colour CCD camera (model DP21) and Cell^B software (Olympus, America).
Photos of malaria parasites observed were captured using a 500 megapixel colour CCD camera (model DP21) and Cell^B software (Olympus, America).
5
Eukitt-Mounted Microscopy Imaging
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Slides were mounted using Eukitt quick-hardening mounting medium (Sigma-Aldrich) and visualized at -0.4°C on a Nikon TiE 3000 inverted microscope (Nikon) equipped with a digital camera (DS-U2/L2-Ri1 digital microscope camera (Nikon) for light microscopy or DXM-1200C coded digital camera (Nikon) for fluorescent microscopy) and Nikon NIS Elements AR software (Nikon). Exposure times were consistent for each staining type. Image levels were equally adjusted using Adobe Photoshop CS6 software (Adobe) to remove nonspecific background staining. Necropsy images were captured on a handheld digital camera and stored as high-resolution JPEG files. Margins of cropped images are indicated by a solid black or white border.
6
Luxol Fast Blue Staining of Brain Sections
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Brain sections (5 μm thick) were deparaffinized in xylene and thereafter hydrated to 5% ethanol. The sections were then incubated in LFB (#26681-01, Electron Microscopy Sciences, USA) solution (0.1% LFB in 95% ethanol, 5% methanol with 0.05% glacial acetic acid) at 56 °C for 16 h. Thereafter, the sections were differentiated with 0.05% lithium carbonate solution (#26681-04, Electron Microscopy Sciences, USA) for 30 s followed by 70% ethanol. The sections were further differentiated in 95% ethanol and rehydrated before mounting with Eukitt® Quick-hardening mounting medium (#03989, Sigma, USA).
7
Fluorescent Microscopy Imaging Protocol
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Slides were mounted using Eukitt Quick-hardening mounting medium (Sigma-Aldrich) and visualized at − 0.4 °C on a Nikon TiE 3000 inverted microscope (Nikon) equipped with a digital camera (DS-U2/L2-Ri1 digital microscope camera (Nikon) for light microscopy or DXM-1200C coded digital camera (Nikon) for fluorescent microscopy), and Nikon NIS Elements AR software (Nikon). Exposure times were consistent for each staining type. Image levels were equally adjusted using Adobe Photoshop CS6 software (Adobe) to remove nonspecific background staining.
8
Histological Analysis of Mouse Eyes
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Mouse eyes from postnatal stages were analyzed histologically for eye pathologies. Tissues were fixed in Davidson solution and embedded in JB-4 plastic medium (Polyscience, Inc., Eppelheim, Germany) according to the manufacturer's protocol. Sectioning was performed with an ultramicrotome (OMU3; Reichert-Jung, Walldorf, Germany). Serial transverse 3-µm sections were cut with a glass knife and stained with methylene blue and basic fuchsin. The sections were evaluated with a light microscope (Axioplan; Carl Zeiss, Jena, Germany). Images were acquired by means of a scanning camera (AxioCam; Jenoptik, Jena, Germany).
For transmission electron microscopic analysis, retinal fragments were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde, postfixed in 1% osmium tetroxide, stained in aqueous uranyl acetate, dehydrated, and embedded in epoxy resin. Ultrathin sections (70 nm) were stained using lead citrate and examined by a transmission electron microscope (Hitachi H-7000; Hitachi, Tokyo, Japan).
The cross sections (2–3 µm) of the optic nerve were immersed in 1% toluidine blue solution and dried on a hot plate for 1 minute, followed by washing them with tap water. The slides were mounted with EUKITT quick-hardening mounting medium (Sigma-Aldrich Chemie GmbH, Munich, Germany). The number of total axons was counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
For transmission electron microscopic analysis, retinal fragments were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde, postfixed in 1% osmium tetroxide, stained in aqueous uranyl acetate, dehydrated, and embedded in epoxy resin. Ultrathin sections (70 nm) were stained using lead citrate and examined by a transmission electron microscope (Hitachi H-7000; Hitachi, Tokyo, Japan).
The cross sections (2–3 µm) of the optic nerve were immersed in 1% toluidine blue solution and dried on a hot plate for 1 minute, followed by washing them with tap water. The slides were mounted with EUKITT quick-hardening mounting medium (Sigma-Aldrich Chemie GmbH, Munich, Germany). The number of total axons was counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
9
Immunohistochemical Detection of ALK and p-ALK
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TMA sections were deparaffinized by using a Tissue-Tek® DRS™ instrument (Sakura). Endogenous peroxidase was blocked by immersion in 0,85% H2O2 (35% Arcos organics, the Netherlands) for 30 minutes at room temperature. ALK Antigen retrieval was performed by heating slides for 10 minutes in a target retrieval solution (EnVision™ FLEX Target Retrieval Solution HIGH pH, Denmark) and for p-ALK 15 min (EnVision™ FLEX Target Retrieval Solution LOW pH, Denmark) using a pressure cooker (decloaking chamber, Biocare Medical). ALK expression was detected using a rabbit monoclonal antibody (clone D5F3®, Cell Signaling Technology; dilution 1:150,) and the presence of p-ALK was detected using a monoclonal rabbit antibody (Tyr1604, Cell Signaling technology; dilution 1:25). Antibodies were diluted in a Normal antibody diluent (Normal antibody diluent, ImmunoLogic, the Netherlands). Sections were incubated with the primary antibodies overnight at +4°C. Antibodies were detected using BrightVision polyHRP-Anti-Rabbit IgG (ImmunoLogic, Amsterdam, The Netherlands) and immunostained using Vector laboratories ImmPACT[R] DAB Substrate Kit, Peroxidase (dilution 1 drop per 1 ml) for 5 minutes at room temperature. Finally, slides were counterstained using Mayers Hematoxylin (Lillie´s Modificatin, Dako, USA) and mounted (Eukitt® Quick-hardening mounting medium, SIGMA-ALDRICH®, Germany).
10
Tissue Staining Protocol Using Iron Hematoxylin
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