Bond max ihc stainer

Samples from patients treated at Vanderbilt University Medical Center for primary (n = 115) and metastatic (n = 111) colon cancer along with adjacent and matched normal tissues were assembled into tumor microarrays by a GI pathologist (M. Kay Washington, M.D.). Immunohistochemistry for ASCT2 was carried out on 5-μm sections of formalin-fixed, paraffin-embedded tissue using the avidin-biotin complex method using the Vectastain Elite ABC kit (Vector Laboratories). Briefly, slides were placed on a Leica Bond Max IHC stainer and heat-induced antigen retrieval was performed using an Epitope Retrieval 2 solution for 20 min. Subsequently, the slides were placed in a Protein Block (×0909, DAKO) for 10 min and then incubated with anti-SLC1A5 (ASCT2; HPA035240, Sigma) for 1 h at a 1:5000 dilution. A Bond Polymer Refine detection system was used for visualization. Of the samples obtained from 115 patients with primary colon cancers and matching normal tissues and 111 metastatic colon cancers and matched normals, a somewhat smaller number contained sufficient quantities of evaluable tumor or matched normal tissue (n = 97 primary tumors plus normal, n = 84 metastatic tumors plus normal). ASCT2 levels were objectively quantitated in a blinded manner as the mean score of three independent reviewers on a scale from 0 (lowest immunoreactivity) to 3 (highest immunoreactivity).

IHC was performed with heat-induced antigen retrieval on the Leica Bond Max IHC stainer using the Epitope Retrieval 2 solution. Antibodies included rabbit anti-human c-MYC monoclonal (Y69, ab32072, Abcam Laboratories) (20 min retrieval, 1 hr incubation with 1:600 dilution); mouse anti-human p53 monoclonal (DO-7, Leica Biosystems) (30 min retrieval, 30 min incubation with ready to use (RTU) antibody); mouse anti-human BCL2 monoclonal (124, bcl-2-100-D5, Leica) (30 min retrieval, 15 min incubation with RTU antibody); and mouse anti-human BCL6 monoclonal (LN22, PA0204, Leica) (10 min retrieval, 15 minute incubation with RTU antibody). The Leica Bond Polymer Refine Detection (DAB) system was used for visualization. Samples were considered positive if >25% of tumor cells were positive for the antibody, except for MYC for which >40% positivity is the standard cut-off [18 (link)].

Slides were placed on the Leica Bond Max IHC Stainer. All steps besides dehydration, clearing, and cover slipping were performed on the Bond Max. Slides were de-paraffinized. Heat induced antigen retrieval was performed on the Bond Max using their Epitope Retrieval 2 solution for 20 minutes. Slides were incubated with anti-FXRl (Catalog # - HPA018246, Sigma, St. Louis, MO) at a dilution of 1:250 for 60 minutes. The Bond Polymer Refine detection system was used for visualization. Slides were then dehydrated, cleared and cover slipped. Staining was scored as absent (0), weak (1), or strong (2) by a pediatric pathologist who was blinded to all other data.

Slide preparation and staining was completed by the Vanderbilt Translational Pathology Shared Resource Core Facility. Paraffin-embedded hemi-brains were sectioned at 5 micrometers. Sections were deparaffinized in xylene, 100% and 70% ethanol and distilled water. Slides were placed on the Leica Bond Max IHC stainer. Heat induced antigen retrieval was performed using Epitope Retrieval 2 solution for 15 minutes. Slides were placed in a Protein Block (Ref# x0909, DAKO) for 10 minutes. Slides were incubated with cleaved Caspase-3 (Cat. 9664, Cell Signaling, Danvers, MA; 1:300 or anti-GFAP (Cat.# ab16997, abcam, Cambridge, MA; 1:500 dilution) for one. The Bond Polymer Refine detection system was used for visualization. Slides were the dehydrated, cleared and cover-slipped. For Flurojade C staining 10 micrometer sections were cut from the same paraffin blocks, and deparaffinized as above. Sections were treated with 0.06% potassium permanganate for 15 minutes to suppress non-specific fluorescence. Slides were stained with 0.001% FluroJade performed for 30 mins in a light-tight box, followed by washing in distilled water. Once dry, sections were cover-slipped using Dapi Mounting media.

Immunohistochemical (IHC) analyses were performed using the Leica Bond Max IHC stainer. All steps besides dehydration, clearing and cover-slipping were performed on the Bond Max. Slides were deparaffinized and heat-induced antigen retrieval was performed on the Bond Max using their Epitope Retrieval 2 solution for 30 minutes. The sections were incubated with Ready-to-Use antibody as indicated below. The Bond Refine Polymer detection system was used for visualization. Slides were then dehydrated, cleared and coverslipped. IHC slides were scanned at the Digital Pathology Shared resource. The automated quantification of the percentages of the KI67-positive cells was performed by Leica Biosystems’ Digital Image Hub, using the software available with the Leica SCN400 Slide Scanner. The antibodies used were as follows: anti-p53 (PA0057, Leica Biosystems, Newcastle, United Kingdom) for 30 minutes; anti-MelanA (PA0233, Leica, Buffalo Grove, IL) for 15 minutes; anti-Ki67 (RM47–26, StatLab, McKinney, TX) for 30 minutes; anti-SOX-10 (PA0813, Cell Marque, Rocklin, CA) for one hour.