Vector novared substrate

PBMCs of the four donors were assayed in an ELISPOT assay for IFN-γ production as previously described (17 (link)). The cells were stimulated with increasing concentrations of 10-mer ¹³⁹TMFPHIIVDV¹⁴⁸ peptide (0.05, 0.1, 0.5, 1, 2, and 4 μg/ml final concentration), 4 μg/ml of 18-mer peptide 99-20, 4 μg/ml irrelevant 9-mer peptide (negative control) or 50 μg/ml of phytohemagglutinin (PHA, positive control). Briefly, ninety-six-well nitrocellulose filter plates (Millipore) were coated with anti-human IFN-γ capture antibody (Mabtech) and blocked with 10% FBS in CM. PBMCs (0.2 × 10⁶ cells/well) were plated with stimulants and incubated for 20 h at +37°C and 5% CO₂. Biotinylated anti-human IFN-γ antibody (Mabtech) followed by streptavidin-HRP (BD, New Jersey, United States) was used for detection. The spots were developed with Vector Nova Red substrate (Vector Labs, Burlingame, United States) and the plates were analyzed using ImmunoSpot Series II analyzer (CTL Europe, Leinfelden-Echterdingen, Germany). The results are expressed as mean spot forming cells (SFC)/10⁶ PBMCs of the duplicate wells.

Lungs harvested after 3 h and 6 h were fixed in 4% paraformaldehyde for 48 h and embedded in paraffin. For morphological analysis of lung injury, paraffin-embedded lung sections harvested 3 h after surgery were stained with hematoxylin and eosin. Neutrophil granulocytes and mucosal mast cells were stained using Ly6G antibody (1:200, BioLegend, CA, USA) and Mcpt-1 antibody (1:100, Invitrogen, MA, USA), 3 h and 6 h after surgery, respectively. To this end, lung sections were deparaffinized in xylene, rehydrated in descending ethanol gradient, and boiled in sodium citrate buffer for antigen retrieval. Sections were blocked with 5% goat serum for 1h at RT and incubated with the primary antibodies. Species-specific IgG was used as negative control for Ly6G staining and rabbit serum for Mcpt-1 staining. As secondary antibody, goat anti-rat IgG-biotin (1:200 and 1:100 (Life Technologies, Carlsbad, CA, USA), respectively, for Mcpt-1 and Ly6G staining) was used and incubated at RT for 1 h (Mcpt-1) or 30 min (Ly6G). For signal detection, the Vectastain Elite ABC kit and Vector NovaRed substrate (both Vector laboratories Inc., Burlingame, USA) were used according to the manufacturer’s instructions. Sections were counterstained with hematoxylin and analyzed by light microscopy (Leica DMI600B, Leica, Wetzlar, Germany).

Tissue sections from the TMA were deparaffinized, rehydrated through graded alcohols, and washed with phosphate-buffered saline. Antigen retrieval was performed by heating sections in 10 mM citrate (pH 6.0) boiling buffer for 15 min. The tissues were permeabilized with 0.3% Triton X for 1 h at room temperature. After endogenous peroxidase activity was quenched and nonspecific binding was blocked, mouse monoclonal anti-Claudin-2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-VDR (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-TGR5 antibodies (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated at 4 °C overnight. Biotinylated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was allowed to incubate for 1 h. After washing, sections were incubated with avidin-biotin–peroxidase complex (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. For color reaction development, slides were immersed in Vector NovaRed substrate (Vector Laboratories, Burlingame, CA) for 2 min and counterstained with Flex Hematoxylin for 2 min (Vector Laboratories, Burlingame, CA). A negative control was performed by replacing anti-VDR antibody with normal serum.