Anti cytokeratin 5 antibody

ExoQuick Plasma prep and Exosome precipitation kit were purchased from System Biosciences Inc. (Palo Alto, California, USA), MiRCURY™ Exosome Isolation Kit-Serum and Plasma were purchased from EXIQON (Woburn, MA, USA), SDS-PAGE Sample Loading Buffer 5X, Bradford Protein Assay kit and Nitrocellulose membrane were purchased from Beyotime Inc. (Shanghai, P. R. China). Precision Plus Protein™ Kaleidoscope™ Standards, Precision Protein™ StrepTactin-HRP Conjugated and Precision Protein StrepTactin-AP Conjugate were purchased from BIO-RAD Inc. (Hercules, California, USA), ExpressPlus™ PAGE Gels, Tris-MOPS-SDS Running Buffer Powder and Transfer Buffer Powder were purchased from GenScrit Inc. (Nanjing, Jiangsu, P. R. China), Stripping Buffer was purchased from CWBio Inc. (Shanghai, P. R. China). The CD63 rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA). CEA monoclonal antibody, Pierce Goat Anti-Rabbit IgG, (H + L), Peroxidase Conjugated and ECL buffer were purchased from ThermoFisher Scientific Inc. (Rockford, IL, USA), anti-Cytokeratin 5 antibody, anti-HE4 antibody, Anti-MUC1 antibody, anti-alpha 1 Fetoprotein antibody, anti-CA19–9 antibody and anti-Grp75 (mortalin) antibody were purchased from Abcam Inc. (Cambridge, MA, USA).

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 carried out to evaluate epidermal differentiation in the 3D bio-printed constructs using anti-loricrin antibody (Abcam, dilution 1:200,) and anti-cytokeratin 5 antibody (Abcam, dilution1:200) referring to the previous protocol [18 (link)]. Briefly, paraffin-embedded samples were cut into 7 μm sections. After dewaxing and rehydration, samples were incubated overnight with the primary antibodies at 4°C. Following this, secondary Alexa Fluor 488 and Alexa Fluor 594-conjugated anti-mouse or anti-rabbit antibodies were added and incubated for 1 h at room temperature. Nuclear counterstaining using DAPI (Sangon Biotech, China) was carried out routinely. Images were acquired using CQ1 (YOKOGAWA, Tokyo) high-content microscope.