I6000

Biogenex I6000 automated immunostainer was used to carry out IHC on formalin fixed paraffin embedded xenografts. Antigen was retrieved by steaming with Cell Marque Declere reagent. Background was blocked with Biogenex Power block. Primary antibody was applied for 1 hour at room temperature followed by detection with the two-step, Hi-Def Polymer Detection kit from Cell Marque, followed by Cell Marque DAB chromagen. Samples were counterstained with hematoxylin, dehydrated, cleared, and coverslipped. We used the following primary antibodies: p21 (Abcam) and Ki67 (Cell Marque). The Millipore Apoptosis kit was used according to manufacturers’ protocol.

A tissue microarray (TMA) was constructed with tumor samples collected from 22Rv1 mouse prostate cancer model. Paraffin sections of the TMA were processed and stained with antibodies using a Biogenex I6000 automated stainer. Digital images of the slides were obtained through an Aperio AT Turbo scanner at 20×. The following antibodies were used for immunohistochemistry staining: Cleaved Caspase 3 (Cell Signaling Technology, 9661), p21 (Cell Signaling Technology, 2947), APC (Abcam, ab15270), Ki67 (Cell Marque, 275R-18), Rb (Abcam, ab181616), p53 (Santa Cruz, sc-126), SMAD4 (Santa Cruz, sc-7966), AR (Abcam, ab105225, and ARv (Abcam, ab198394).

Immunohistochemistry was performed as previously described [25 (link)]. Briefly, five-micrometer sections were deparaffinized in xylene and rehydrated in graded ethanol. For antigen retrieval, sections were microwaved in citrate buffer pH 6.0 (BioGenex, San Ramon, CA, USA) for 12 min at 95°C and cooled for 30 min prior to immunostaining. Sections were incubated with 3% hydrogen peroxide for 15 min, followed by incubation with primary antibody for 60 min. An automated immunostainer (i6000; BioGenex) was utilized for subsequent incubation steps: sections were incubated in MultiLink biotinylated anti-IgG for 20 min, horseradish peroxidase conjugated secondary antibody for 20 min, followed by development with 3-amino-9-ethyl-carbazole for 10 min (BioGenex). Sections were then counterstained with hematoxylin. All incubation steps were performed at room temperature, and sections were washed with Tris-buffered saline between incubations.

Formalin-fixed paraffin-embedded (FFPE) tissue blocks were sectioned at 4 μm, and deparaffinized through three washes in xylene and a decreasing series of ethanol. Antigen retrieval was performed in a steam cooker for 15 minutes in Declere (Cell Marque) working solution. Endogenous hydrogenase was blocked with 3% hydrogen peroxide for five minutes. Slides were incubated in casein-based protein block (Biogenex) for 20 minutes before incubation with the respective antibodies at room temperature for 30 minutes. Slides were then rinsed with buffer and incubated with Amplifier from Hi-Def Polymer Detection Kit (Cell Marque) for 10 minutes at room temperature. Afterwards slides were rinsed with buffer and incubated in DAB chromogen for six minutes at room temperature for color development. The slides were counterstained with Hematoxylin I (Richard Allan Scientific), rinsed in water, and dehydrated through a series of increasing ethanol and three changes of xylene. Slides were mounted on coverslips. Digital images of slides were generated via Aperio AT scanner at 20×. Immunohistochemistry assays were performed on a Biogenex i6000 automated stainer. Masson's Trichrome (Polyscientific) staining was performed manually as per the manufacturer's instructions.