Immunohistochemical Detection of Brucella

Five micrometer tissue sections of testicle/epididymis (mouse and guinea pig) and prostate (guinea pig only) were adhered to positively charged glass slides for immunohistochemistry. Slides were routinely processed, and antigen retrieval was performed as previously described using a 2,100 Antigen Retriever (Aptum Biologics Ltd. Southampton, UK; Hensel et al., 2019 (link)). Slides were blocked as previously described with Bloxall Blocking Solution (Vector Laboratories, Burlingame, CA) and normal goat serum (Vector Laboratories; Hensel et al., 2019 (link)). Primary incubation was performed overnight at 4^o C with a Brucella polyclonal rabbit antibody (Bioss Antibodies, Boston, MA) at dilution of 1:500. A Vectastain Elite® ABC HRP Kit (Vector Laboratories) with an avidin/biotinylated anti-rabbit secondary antibody was used according to the manufacturer’s instructions. Antigen was visualized with a Betazoid DAB chromagen kit (Biocare Medical, Pachecho, CA). The slides were counterstained with Gills’s hematoxylin III and cover slipped.

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Hensel M.E., Stranahan L.W., Edwards J.F, & Arenas-Gamboa A.M. (2022). Intratracheal inoculation results in Brucella-associated reproductive disease in male mouse and guinea pig models of infection. Frontiers in Microbiology, 13, 1029199.

Publication 2022

Bloxall Blocking Solution normal goat serum Brucella polyclonal rabbit antibody Vectastain Elite® ABC HRP Kit Betazoid DAB chromagen kit

Anti antibody Antibodies Antibody Antigen Avidin Biologics Brucella Dilution Epididymis Gills Goat Guinea pig Hematoxylin Immunohistochemistry Mouse Prostate Rabbit Serum Testicle Tissue Vector

Corresponding Organization : Texas A&M University

Other organizations : The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

Tissue sections (mouse and guinea pig testicle/epididymis, guinea pig prostate)

dependent variables

Immunohistochemical staining of Brucella antigen

control variables

Tissue section thickness (5 micrometer)
Slide type (positively charged glass slides)
Antigen retrieval method (2,100 Antigen Retriever, as previously described)
Blocking (Bloxall Blocking Solution and normal goat serum, as previously described)
Primary antibody (Brucella polyclonal rabbit antibody, 1:500 dilution)
Secondary antibody (avidin/biotinylated anti-rabbit from Vectastain Elite® ABC HRP Kit)
Chromagen (Betazoid DAB)
Counterstaining (Gill's hematoxylin III)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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