Mouse monoclonal antibodies

Immunohistochemical staining was performed using a Autostainer Plus (Dako, Hamburg, Germany) and mouse monoclonal antibodies from Dako, Medac (Hamburg, Germany), and Thermo Fisher (Dreieich, Germany) at dilutions shown in Table 1. A polyclonal antibody was only used for myeloperoxidase (MPO) staining. We followed the specific standardized protocol supplied by the manufacturer. Omission of the primary antibody served as a negative control. Briefly, slides were pretreated with Trilogy buffer (Medac; 1:100, 16 min at 95°C), citrate low buffer (Thermo Fisher; 1:100, 26 min at 98°C), or pronase E (Merck; 0.1%, 10 min at room temperature) followed by treatment with 3% H₂O₂ for 8 min. All antibodies were applied in a volume of 200 μl and incubated for 30 min. After washing (Medac wash buffer, 1:20 in aqua dest), secondary antibody (Medac) was applied in a volume of 200 μl and incubated for 20 min. After washing, each sample was incubated with polymer (200 μl) for 30 min (note that the secondary antibody and polymer are components of the color-coded BrightVision HRP kit from Medac). The slides were washed twice and incubated in Bright DAB (Medac) for 10 min. The slides were washed in aqua dest, counterstained with hematoxylin for 8 min, and washed before coverslipping [20 (link), 22 (link)].

Spleen, lung, liver, and lymph nodes were perfused with PBS followed by 4% paraformaldehyde and then post-fixed overnight and processed for paraffin embedding. Five-micron thick sections were cut from the paraffin blocks, mounted on glass slides, and labeled with mouse monoclonal antibodies (Dako) for HLA-DQ/DP/DR (clone CR3/43, 1:100) and HIV-1p24 (1:10). The polymer-based HRP-conjugated anti-mouse Dako EnVision system was used as a secondary detection reagent and developed with 3,3′-diaminobenzidine (DAB). All paraffin-embedded sections were counterstained with Mayer’s hematoxylin. Deletion of primary antibodies or using mouse IgG served as controls. Images were obtained with a Nikon DS-Fi1 camera fixed to a Nikon Eclipse E800 (Nikon Instruments, Melville, NY) using NIS-Elements F 3.0 software.

Spleen, lung, liver, and lymph nodes were perfused with PBS followed by 4% PFA and then postfixed overnight and embedded in paraffin. Five-micron-thick sections were cut from the paraffin blocks, mounted on glass slides, and labeled with mouse monoclonal antibodies (DakoCytomation) for HLA-DQ/DP/DR (clone CR3/43, 1:100) and HIV-1p24 (1:10). The polymer-based HRP-conjugated anti-mouse Dako EnVision system was used as a secondary detection reagent and developed with DAB. All paraffin-embedded sections were counterstained with Mayer’s hematoxylin. Deletion of primary antibodies or mouse IgG served as controls. Images were obtained with a Nikon DS-Fi1 camera fixed to a Nikon Eclipse E800 (Nikon Instruments) using NIS-Elements F 3.0 software.

p21, p53, and c-myc were detected by immunohistochemical method. All primary antibodies and mouse monoclonal antibodies were purchased from Dako (Hamburg. Germany). Immunohistochemical staining was performed by the enhance labeled polymer system (ELPS) method. After overnight incubation at 4°C with anti-p21, p53, and c-myc antibody, sections were treated according to standard immunoperoxidase methods using a streptavidin biotin peroxidise complex kit (LSAB + Kit/HRP; Dako). The peroxidise reaction was then developed with diaminobenzidine (Dako). Negative control sections were subjected to the same procedure except that the first antibody was replaced by phosphate buffer saline (PBS) [20 (link)].