Opal 4 color ihc kit

Human subject protocol (2007-0511) was approved by Institutional Research Board at M.D. Anderson Cancer Center. Tissue microarrays (TMA) were generated using a Beecher instrument with 0.6 mm cores taken from the donor block and placed into the recipient block in triplicate for each case. Tissue microarrays with triplicate cores for each case were generated from primary RCC. Murine tumor TMA were generated with 5 mm cores. TMAs were immunohistochemically stained and analyzed using inForm software (Caliper Life Sciences). The slides were stained using Opal 4-color IHC Kit (NEL794001KT) from Perkin Elmer. Microwave treatment (MWT) was applied to perform antigen retrieval, quench endogenous peroxidases, and remove antibodies from earlier staining procedures. Perkin Elmer AR6 Antigen retrieval buffer (pH 6) was used for CD8 and PD-1 staining while Perkin Elmer AR9 Antigen Retrieval buffer (pH 9) was used for P-STAT1 staining. The slides were stained with primary antibodies against CD8 and PD-1, corresponding HRP conjugated secondary antibodies, and subsequently TSA dyes to generate Opal signal (CD8, Opal 520; PD-1, Opal 570, or Opal 620). The slides were scanned with the Vectra image scanning system (Caliper Life Sciences), and signals were unmixed and reconstructed into a composite image with Vectra inForm software 2.4.6.

Paraffin-section containing pancreas tissue were deparaffinized, dehydrated, and boiled for epitope retrieval using an antigen retrieval buffer at pH = 6.0 (Opal 4-color IHC Kit, PerkinElmer, Waltham, MA). Tissue sections were blocked for 10 min and incubated with primary antibodies overnight at 4°C. Primary antibodies include: MAFA, PDX1 (Cell Signaling, Danvers, MA, USA), pancreatic polypeptide, somatostatin, NKX2.2, PAX4, PAX6, ARX, MAFA (Santa Cruz Biotechnology, Dallas, TX, USA), Ki67, PDX1 (Abcam, Cambridge, UK), glucagon and insulin (Sigma-Aldrich, St. Louis, MO, USA). The sections were incubated with Polymer HRP- conjugated secondary antibodies for 10 min at room temperature. The primary antibodies were stained with repeat procedures including antigen stripping and blocking steps. Cell nuclei was stained via adding mounting media containing fluoroshield with DAPI. The images were visualized by confocal microscopy (C1-Si, Nikon). To quantify colocalization, we used the Pearson's correlation coefficient (R_r). The images were analyzed by using PSC Colocalization plug-in (ImageJ) 25 (link), 26 (link). R ranges between -1 (perfect negative correlation) to +1 (perfect positive correlation) with 0 meaning no correlation.

Tissue was fixed overnight in 4% PFA, cryoprotected in 30% sucrose/PBS, and embedded in Tissue-Tek O.C.T. (Sakura Finetek) prior to cutting 6  μm cryosections on Superfrost Plus slides (VWR). Cultured cells grown on coverslips were fixed for 20 min in 4% PFA then rinsed in PBS. Permeabilization was performed with 1 × TBST (Tris-buffered saline: 25 mm Tris, 0.14 m NaCl, 0.1% Triton X-100) for 15 min at room temperature (RT). Three percent% goat or horse serum in TBST × 30 min was used for block. Primary antibodies applied for 1 h at RT or O/N at 4 °C in block. Alexa Fluor secondary antibodies were applied at 1:500 dilution for 1 h RT. Nuclei were counterstained in 4′,6-diamidino-2-phenylindole (DAPI; Santa Cruz) and mounted with FluorSave Reagent (Calbiochem). For immunostaining of tissue sections when two or more primary antibodies were from the same host species and for comparison of cell type specific CIC expression, the Opal 4-color IHC kit (Perkin Elmer) was used per manufacturer’s protocol.