Polymer hrp ihc detection system

Patient specimens (n = 3) were deparaffinized, rehydrated and subjected to antigen retrieval by boiling in a retrieval buffer for 15 min. The sections were cooled in PBS for 10 min. The samples were blocked in 5 mg/mL BSA for 1 h before hybridizing with 1:200 diluted primary antibodies against MSI1 and CD163 at 4 °C overnight, and then Alexa Fluor 488- or 555-conjugated secondary antibodies were added at 1:200 dilution and the samples were incubated at room temperature for 1 h. The sections were then washed twice in PBS and stained with DAPI. Sample slices were flat-mounted with a mounting solution. Immunohistochemistry (IHC) tumor specimens from mice were fixed with 4% paraformaldehyde. The sections were deparaffinized, rehydrated and subjected to antigen retrieval by boiling in a retrieval buffer for 15 min. The sections were cooled in PBS for 10 min. The samples were blocked in 5 mg/mL BSA for 1 h before hybridizing with 1:150 diluted primary antibodies (MIF1, MSI1 and CD206) at 4 °C overnight. The signals were amplified by a Polymer-HRP IHC Detection system (BioGenex, San Ramon, CA, USA) following the manufacturer’s instructions. The sections were examined under an Olympus BX61 microscope.

Human clinical breast cancer specimens were obtained from patients at the time of initial surgery and were fully encoded to protect patient confidentiality. Clinical specimens were utilized under a protocol approved by the Institutional Review Board of the Human Subjects Research Ethics Committee of Chang Gung Memorial Hospital. For GHCer staining, tissue sections were deparaffinized followed by antigen retrieval by autoclaving at 121 °C for 5 min in AR-10 solution (Biogenex, Fremont, CA, USA). Slides were incubated with mAb VK9 (1:100 antibody dilution buffer) (Ventana Medical Systems, Inc., Oro Valley, AZ, USA) overnight at 4 °C, followed by antibody detection using a polymer-HRP IHC detection system (Biogenex). The slides were counter stained with hematoxylin and mounted. Digital images were captured using an Aperio ScanScope XT Slide Scanner (Aperio Technologies, Vista, CA, USA) under 20× magnification. The expression of Globo H and the morphology of tumor blood vessels were confirmed by pathologists.