Immunohistochemistry was performed on slices with a 4 µm thickness of paraffin-embedded samples, as already described33 (link)–35 (link). The primary antibody Pan-granzyme (isotype Goat polyclonal IgG, SC-1969; Santa Cruz Biotechnology, Dallas, TX, USA) was utilized at 1:50 dilution, and the detection system was LSAB + System-HRP (K0690; DakoCytomation, Carpinteria, CA, USA); NCAM-1/CD56 (isotype Mouse monoclonal IgG1, ab200698; Abcam, Cambridge, MA, USA) was utilized at 1:100 dilution, and the detection system was Novolink Max Polymer Detection System (RE7280-K; Leica Biosystems, Newcastle Upon Tine, UK), and the chromogen used was DAB (3,3′ diaminobenzidine, D5637, Sigma). We performed scanning of immunohistochemically stained specimens using Aperio Scan-scope Cs (Aperio Technologies, Vista, CA, USA). Images of the scanned stained specimens were analyzed utilizing the software Image-Pro Plus version 4.5.0.29 (Media Cybernetics Inc., Bethesda, Maryland, USA)34 (link),36 (link). Using the software, we obtained the calculation of the total tissue distribution of Pan-granzyme and NCAM-1/CD56 dividing the total number of doubly stained cells by the total area measured in the dermis. The expression of the measured markers was normalized by the total area and expressed in cells/μm2.
Lsab system hrp k0690
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The LSAB + System-HRP (K0690) is a laboratory equipment product manufactured by Agilent Technologies. It is designed for use in immunohistochemistry (IHC) procedures. The product incorporates a labeled streptavidin-biotin (LSAB) detection system with a horseradish peroxidase (HRP) label. This system is used to visualize target antigens in tissue samples.
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2 protocols using lsab system hrp k0690
1
Immunohistochemical Analysis of Granzyme and NCAM-1/CD56
2
Immunohistochemical Analysis of Toxic Hepatopathy
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Archival (1998-2012) paraffin-embedded samples of the telencephalon, brainstem, cerebellum, and liver from 22 cattle that died with acute toxic hepatopathy were selected, sectioned at 3 µm, and stained with hematoxylin and eosin and periodic acid-Schiff. Immunostaining with anti-GFAP (polyclonal rabbit anti-GFAP antibody, Z0334, Dako Denmark, Glostrup, Denmark), anti-S100 protein (polyclonal rabbit anti-S100 protein antibody, Z0311, Dako Denmark), and anti-vimentin (monoclonal mouse anti-vimentin antibody, Invitrogen, Carlsbad, CA) antibodies was also performed, using the biotin-peroxidase-streptavidin method (LSAB+ system-HRP, K0690, Dako Denmark), with DAB (Dako Denmark) or NovaRed (Vector Red S-K 4800, Vector Laboratories, Burlingame, CA) as chromogen (Table 1).
Additional information was retrieved from the files. The brain specimens from 11 adult bovids that died from various causes without neurologic clinical signs or hepatic and/or brain changes served as negative controls.
Additional information was retrieved from the files. The brain specimens from 11 adult bovids that died from various causes without neurologic clinical signs or hepatic and/or brain changes served as negative controls.
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