Bond antibody diluent buffer

Immunohistochemical staining performed by Leica Biosystems BOND RX for formalin-fixed, paraffin-embedded and cut in 3 μm thick tongue sections according to optimized (for the specific tissue) standard protocols. Histological preparation procedures have been reported in detail previously [21 (link)]. Stainings comprised 30 min incubation at 95 °C, dewaxing with Leica Bond Dewax solution (Leica-Biosystems, Inc., Buffoalo. Groove, IL; Cat.no. AR9222), antigen retrieval with Bond Epitope Retrieval‑1 solution (Leica Biosystems, Inc., Buffoalo. Groove, IL; Cat. no. AR9961), and blocking of unspecific binding sites with 2% goat serum. Primary antibody binding was visualized with diaminobenzidine chromogen and a hematoxylin counterstain, using the Leica Bond Refine Detection kit (Leica-Biosystems, Inc., Buffalo. Groove, IL; Cat. no. DS9800). Primary antibodies were diluted in the Leica Bond Antibody Diluent buffer (Leica-Biosystems, Inc., Buffoalo. Groove, IL; USA, Cat. no. AR9352) as follows [20 (link)]; anti-p16 ARC antibody 1:50 (Abcam, Cambridge, MA, USA; Cat.no. 51243; rabbit-monoclonal), anti-p21 Ras antibody 1:100 (Abcam, Cambridge, MA, USA; Cat.no. 86696; mouse-monoclonal), and anti-cytokeratin 5 antibody 1:5000 (Abcam, Cambridge, MA, USA; Cat.no. 52635; rabbit monoclonal).

IHC was conducted on 1 µm thick full tissue sections and stained with markers for tumor-infiltrating lymphocytes (TILs; CD3, CD8) and immune checkpoint proteins (PD-1, PD-L1). Staining was performed using a Leica Bond RX Automated Stainer (Leica Products/Equipment, Leica Microsystems, Inc., Buffalo Groove, IL). Slides with incubated for 30 minutes at 95°C, followed by dewaxing with Leica Bond Dewax solution (Leica Biosystems, Inc., Buffalo Groove, IL; Cat no. AR9222), antigen retrieval with Bond Epitope Retrieval 1 solution (Leica Biosystems, Inc., Buffalo Groove, IL; Cat no. AR9961) and blocking of unspecific binding sites with 2% goat serum. Primary antibody binding was visualized with diaminobenzidine chromogen and a hematoxylin counterstain, using the Leica Bond Refine Detection kit (Leica Biosystems, Inc., Buffalo Groove, IL; Cat no. DS9800). Primary antibodies were diluted in Leica Bond Antibody Diluent buffer (Leica Biosystems, Inc., Buffalo Groove, IL; Cat no. AR9352) as follows: anti-CD3 (Abcam, Cambridge, MA, USA; Cat no. ab5690; rabbit polyclonal) 1:300, anti-CD8 (Abcam, Cambridge, MA, USA; Cat no. ab4055; rabbit polyclonal) 1:300), anti-PD-1 (Abcam, Cambridge, MA, USA; Cat no. ab137132; rabbit polyclonal) 1:75, anti-PD-L1 (Abcam, Cambridge, MA, USA; Cat no. ab205921; rabbit polyclonal) 1:100).

Histological preparation procedures have been reported in detail previously [26 (link)]. Stainings comprised 30 min incubation at 95 °C, dewaxing with Leica Bond Dewax solution (Leica Biosystems, Inc., Buffoalo Groove, IL; Cat no. AR9222), antigen retrieval with Bond Epitope Retrieval 1 solution (Leica Biosystems, Inc., Buffoalo Groove, IL; Cat no. AR9961) and blocking of unspecific binding sites with 2% goat serum. Primary antibody binding was visualized with diaminobenzidine chromogen and a haematoxylin counterstain, using the Leica Bond Refine Detection kit (Leica Biosystems, Inc., Buffoalo Groove, IL; Cat no. DS9800). Primary antibodies were diluted in the Leica Bond Antibody Diluent buffer (Leica Biosystems, Inc., Buffoalo Groove, IL; USA, Cat no. AR9352) as follows: anti-claudin-1 1:200 (Abcam, Cambridge, MA, USA; Cat no. 15098; rabbit polyclonal), anti-occludin 1:200 (Abcam, Cambridge, MA, USA; Cat no. 222691; rabbit polyclonal), anti-E-cadherin 1:3000 (Abcam, Cambridge, MA, USA; Cat no. 76055; rabbit polyclonal) and anti-β-catenin 1:700 (Abcam, Cambridge, MA, USA; Cat no. 2365; rabbit polyclonal).