Envision g 2 system ap rabbit mouse

Materials were from Sigma Aldrich (St. Louis, Mo, USA) except rK39 immunochromatographic test strips (InBios International, Seattle, WA, USA), anti-human CD4 (clone 4B12, Novocastra, Newcastle upon Tyne, UK,), Chemokine Receptor 4 (CCR4, clone 1G1), CD3 Peridinin chlorophyll (PerCP, clone SK7), CD8 Alexa Fluor 488 (clone RPA-T8), CD8 PerCP (clone SK1), CCR4 Phycoerythrin (PE, clone 1G1, BD Biosciences, San Jose, CA, USA), PD-1(clone EH12.2H7) and CD127 (clone A019D5) APC (Biolegend, San Diego, CA, USA), IL-5 (clone 9906, R&D Systems, Minneapolis, MN, USA), CCL17 (clone N-20), IL-10 (clone E-10), PD-1 (clone C-20), Perforin H (clone H-315), Granzyme B (clone 2C5, Santa Cruz Biotechnology, Dallas, TX, USA), antibody diluent, Target Retrieval Solution Citrate pH 6 (S2369), secondary detection system EnVision™ G|2 System/AP-Rabbit/Mouse (Permanent Red), EnVision™ FLEX Target Retrieval Solution, EnVision™ DuoFLEX Doublestain System (Dako, Glostrup, Denmark), RNALater and RNAqueous kit (Ambion, Austin, TX, USA), One-step reverse transcription kit from Qiagen (Hilden, Germany) and Bio-Plex Pro™ Human Chemokine Panel 40-Plex (BioRad, Hercules, CA, USA).

Adjacent sections of pancreas were stained for (1) Ki67, insulin and glucagon and (2) insulin, somatostatin and pancreatic polypeptide. Primary antibodies used were mouse anti-Ki67 (1:50; Dako M7240; Carpinteria, CA), guinea pig anti-insulin (1:1,000; Dako A0564), rabbit anti-somatostatin (1:1,000; Dako A056601-2), mouse anti-glucagon (1:1,000; Abcam ab10988, Cambridge, MA;), rabbit anti-pancreatic polypeptide (1:800; Abcam ab113694). Insulin was developed using EnVision™ G|2 System/AP, Rabbit/Mouse (Dako K5355Ki67); Ki67 and somatostatin were developed using EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse, HRP (Dako K406511); glucagon and pancreatic polypeptide were developed using ImmPRESS™ HRP Universal Antibody (Anti-Mouse IgG/Anti-Rabbit IgG, Peroxidase) Polymer Detection Kit (Vector Laboratories MP-7500, Burlingame, CA) with HIGHDEF blue IHC chromogen (HRP) for color development (Enzo Life Sciences ADI-950-151-0030, Farmingdale, NY). Slides were counterstained with hematoxylin (1:10; Dako S330930-2).

Paraffin-embedded skin biopsies were cut into 2 to 5-μm sections, rehydrated, and boiled in sodium citrate buffer (pH 6.0). Sections were stained with mouse anti-MxA (provided by O. Haller, Freiburg University, Breisgau, Germany; 1:400 dilution) followed by staining with EnVision G | 2 System/AP Rabbit/Mouse (Dako) or antibodies against LPAC (Merck ABN2258, 1:15000) and QAGR (Merck ABN2271, 1:20000). Sections were counterstained with Mayer’s hematoxylin (Merck).