Paraffin‐embedded spinal cord sections were immunostained for ET‐1. Native slides were deparaffinized using xylene, rehydrated in graded ethanol, pretreated with heat‐induced antigen retrieval in citrate buffer (pH = 6) at 95°C for 20 minutes using a laboratory microwave. Sections were blocked with 10% normal goat serum for 2 hours at room temperature (RT), and then incubated with antibodies against ET‐1 (monoclonal mouse; 1 : 350 dilution; Abcam; the antibody had been predicted by the provider to bind to canine ET‐1) for 12 hours at 4°C. Subsequently, endogenous peroxidase was blocked with 3% H2O2 in methanol over 15 minutes at RT. Then, slides were incubated with biotinylated goat anti‐mouse IgG (1 : 1000, Milan Analytica) for 1 hour, and thereafter with peroxidase‐streptavidin (1 : 1000, Milan Analytica) for 30 minutes at RT. Immunohistochemical staining was visualized by incubating the slides with 3‐amino‐9‐ethylcarbazole for 6–8 minutes at RT, and by counterstaining with Mayer's hematoxylin. Sections from canine liver tissue treated in the same way served as positive controls. Negative controls were performed using nonspecific mouse IgG instead of the primary antibody in the same concentration on both canine liver tissue and on spinal cord sections.
Monoclonal mouse
Manufactured by Abcam
Sourced in United Kingdom
Monoclonal mouse is a laboratory-generated mouse that produces a single type of antibody. It is a widely used tool in immunological research and diagnostic applications.
Automatically generated - may contain errors
2 protocols using monoclonal mouse
1
Immunohistochemical Detection of ET-1 in Canine Spinal Cord
2
Immunohistochemical Analysis of GFAP and CHI3L1
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GFAP and CHI3L1 expression was studied in the brain of 7 CSVD and 5 control cases without neurological disorders (Table 2 ). The brains of all autopsied subjects were fixed in a 4% formaldehyde solution and underwent a routine neuropathological examination, as described previously [34 (link),35 (link)]. A coronal mid-hemispheric block (approx. 1 cm thick) was embedded in polyethylene glycol (PEG 1000, Merck, Carl Roth Ltd., Karlsruhe, Germany). Multiple 100-µm-thick consecutive sections were obtained from each block with the aid of a sliding microtome (Jung, Heidelberg, Germany). Immunohistochemistry (IHC) was performed with primary antibodies against GFAP (1:1000, monoclonal mouse, Abcam, Cambridge, UK) or CHI3L1 (1:750, polyclonal goat, R&D Systems, Exton, PA, USA) and a secondary biotinylated antibody (1:200; 2 h, room temperature, Vector Laboratories, Burlingame, CA, USA). The immunohistochemical reaction was visualised with an avidin–biotin–peroxidase complex (ABC Vectastain Kit, Vector Laboratories, Burlingame, CA, USA) and the chromogen 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma Taufkirchen, Germany), which was intensified using Cobalt(II)Chloride (32 nM) for the labelling of CHI3L1. Omission of the primary antibody resulted in a lack of staining.
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