Tissue sections of anterior and posterior kidneys, liver, and spleen of all 31 brown trout underwent immunohistochemistry to detect T. bryosalmonae antigens. Heat-induced epitope retrieval (microwave pressure cooker for 33 min) with a tris buffer at pH 9.0 was used for antigen demasking, followed by avidin biotin blocking and normal goat serum to block unspecific reactions. After determination of the final dilution, a monoclonal mouse antibody (IgG1, P01, Aquatic Diagnostics Ltd, Scotland) was used as the primary antibody (1:50 in tris-buffered saline). To increase sensitivity a biotinylated goat anti-mouse antibody served as the secondary antibody (Vector, BA-9200, Burlingame, CA, USA). After incubation with avidin-coupled alkaline phosphatase (ABC-AP, Vector, AK-5000, Burlingame, CA, USA), Liquid Permanent Red (K0640, Dako Agilent, Glostrup, Denmark) was used as the chromogen. Positive (PKD-infected fish) and negative (substitution of the primary antibody with an irrelevant mouse IgG) controls were included in each assay.
Ak 5000
Manufactured by Vector Laboratories
Sourced in United States
The AK-5000 is a versatile laboratory instrument designed for various applications. It functions as a multi-purpose analytical tool, capable of performing a range of tests and analyses. The core purpose of the AK-5000 is to provide accurate and reliable data for researchers and scientists in various fields.
Automatically generated - may contain errors
Lab products found in correlation
Ba 4001, by Vector Laboratories (2 mentions)
Gtx 76011, by GeneTex (2 mentions)
Mca1211, by Bio-Rad (2 mentions)
Sk 5100, by Vector Laboratories (2 mentions)
Abc ap, by Vector Laboratories (1 mentions)
Ba 9200, by Vector Laboratories (1 mentions)
Kaiser s glycerol gelatin, by Merck Group (1 mentions)
Ab37415, by Abcam (1 mentions)
Eclipse ni, by Nikon (1 mentions)
Kaiser s glycerin gelatine, by Merck Group (1 mentions)
4 protocols using ak 5000
1
Immunodetection of T. bryosalmonae
2
Immunohistochemical Quantification of Platelets
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Myocardial tissue was embedded with O.C.T., cut into 10-μm-thick sections, and fixed with acetone. For staining of platelets, a rat anti-mouse CD41 antibody was used (GTX 76011, GeneTex, Irvine, CA), and for control purposes, an IgG1 isotype control was used (MCA1211, Serotec, Puchheim, Germany). Secondary staining was performed with a biotinylated rabbit anti-rat IgG (BA-4001, Vector). Alkaline phosphatase and substrate kit (AK-5000 & SK-5100, Vector) and levamisole (X3021, DAKO) were used for detection. Finally, samples were embedded with Kaiser’s glycerol gelatin (Merck, Darmstadt, Germany) until adequate staining.
Platelets were counted in two representative pictures of × 20 magnification out of the inflamed myocardium of two different CD41–stained slices. For counting MPIOs, the inflamed myocardium of 10 representative CD41 stained slices was examined carefully.
Platelets were counted in two representative pictures of × 20 magnification out of the inflamed myocardium of two different CD41–stained slices. For counting MPIOs, the inflamed myocardium of 10 representative CD41 stained slices was examined carefully.
3
Immunohistochemical Analyses of Autoimmune Liver Diseases
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Immunohistochemical analyses of PSC, PBC and AIH explant liver and non-autoimmune liver disease (AILD) control (non-alcoholic fatty liver disease, NAFLD) liver tissues were performed (ethics number PV4081). Clinical parameters are provided in online supplemental table 3 . 5 µm sections of paraffin-embedded tissues were prepared. Tissues were deparaffinised followed by heat antigen retrival at 121°C for 10 min using S1699, pH6 (DAKO). Tissue sections were incubated with anti-HLA-DPA1 (Atlas, HPA017967) or isotype control rabbit poly IgG (abcam, ab37415) over night at 4°C. After washing with TBS-T, secondary staining was performed with goat α-rabbit-Biotin (LS-Bio, LS-C350860) for 30 min at room temperature. For detection, after washing of the slides, ABC-AP (VECTASTAIN, AK-5000) was added for 30 min, followed by Permanent Red staining (Zytomed Systems, ZUC001-125) for 7.5 min at RT. Subsequently, haematoxylin staining was performed. Image acquisition was performed using a Nikon eclipse Ni.
4
Quantifying Pulmonary Embolism and Microparticles
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Platelets were stained using a rat anti-mouse CD41 antibody (GTX 76011, GeneTex, Irvine/CA, USA) or unspecific control IgG1 isotype (MCA1211, Serotec, Puchheim, Germany). For secondary staining a biotinylated rabbit anti-rat IgG (BA-4001, Vector, Burlingame/CA, USA) was used. Afterwards, alkaline phosphatase and substrate kit (AK-5000 & SK-5100; Vector, Burlingame/CA, USA) after levamisol pre-treatment (X3021, DAKO, Hamburg, Germany) was applied for antibody detection. Sections were then embedded in Kaiser’s Glyceringelatine (1092420100, Merck, Hamburg, Germany). Due to their size (1 μm), MPIO beads are visible using 63x magnification without further staining. MPIOs were quantified as mean number of MPIOs of 3 sections per section at 63x magnification.
Histology sections were analyzed using a light microscope (Zeiss optics, Germany). Pulmonary embolism was quantified as mean of 3 representative sections of the according. Pulmonary emboli were graded according to size of obstructed vessels: small vessel (maximum diameter < 150 μm: GI < 50% vessel obstruction, GII > 50% vessel obstruction) or large vessels (maximum diameter > 150 μm: GIII < 50% vessel obstruction, GIV > 50% vessel obstruction).
Histology sections were analyzed using a light microscope (Zeiss optics, Germany). Pulmonary embolism was quantified as mean of 3 representative sections of the according. Pulmonary emboli were graded according to size of obstructed vessels: small vessel (maximum diameter < 150 μm: GI < 50% vessel obstruction, GII > 50% vessel obstruction) or large vessels (maximum diameter > 150 μm: GIII < 50% vessel obstruction, GIV > 50% vessel obstruction).
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