Opal 7 color automation ihc kit

Staining was performed as previously described (Cherry et al., 2016 (link)). Briefly, tissue blocks of cortical samples were taken from Broadman area 8/9 for all cases. Tissues were embedded in paraffin and cut into 20-µm-thick sections. For chromogenic staining, sections were incubated overnight at 4°C with anti-NUP62 antibody. Sections were treated with biotinylated secondary antibodies and labeled with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories). Sections were counterstained with Gill’s hematoxylin (Vector Laboratories, #H-3401). For multiplex immunofluorescent images, sections were incubated overnight at 4°C with anti-NUP62 (BD Transduction Laboratories, #610497) and or anti-pTDP43 (Cosmo Bio, #NC0877946) antibodies. Fluorescent labeling was carried out using the PerkinElmer Opal 7-Color Automation IHC kit (#NEL801001KT), per manufacturer’s instructions. Sections were counterstained with DAPI.

An automated slide staining machine BOND RX (Leica Biosystems, Nußloch, BW, Germany) and an Opal 7-color Automation IHC kit (Perkin Elmer, Waltham, MA, USA) were used for mIHC staining. The following primary antibodies were used: CD68 (1:2000, clone KP-1, Agilent, Tokyo, Japan), CD163 (1:100, clone 10D6, Leica Biosystems), PD-L1 (1:100, clone SP142, abcam, Cambridge, MA, USA), CD20 (1:1, clone L26, Agilent, Tokyo, Japan), HLA class I (1:800, clone EMR8-5, abcam, Cambridge, MA, USA), and pan-cytokeratin (CK) (1:4, clone AE1/AE3, Agilent, Tokyo, Japan). Staining was performed using the Opal 7-color Automation IHC kit and BOND research detection kit (Leica Biosystems Nußloch, BW, Germany), according to the manufacturer’s instructions. Primary antibodies were incubated for 30 min at 25 °C. Slides were mounted using a ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA).