Tdt enzyme

The NE-induced host immune response of inflammatory gene expression was quantified in ileal tissue samples. The total RNA was extracted by the TRIzol method from ileal tissue samples of birds at day 25 as described before [1 (link),3 (link)] and cDNA was prepared using M-MLV (NE Biolabs). The accumulation of the proinflammatory genes of Ifnγ, Mmp9, and Gapdh in the ileum tissue was determined using SYBR Green PCR Master mix (Bio-Rad) on a Bio-Rad 384-well Real-Time PCR System as described before [1 (link)]. The gene expression of the fold-change was calculated using the ΔΔCt method [1 (link),3 (link)] and Gapdh as an internal control.
The degree of ileal cells apoptosis in the intestinal tissue was assessed using the TUNEL assay as described before [1 (link),3 (link)]. The TUNEL assay detected the late phase of cellular apoptosis based on the fragmentation of nucleic acids. In brief, ileal tissue slides were deparaffinized three times with xylene wash and then rehydrated with 100%, 95%, and 70% ethanol. The tissues were then incubated at 37 °C for 90 min with the TUNEL solution (5 μM Fluorescein-12-dUTP (Enzo Life Sciences), 10 μM dATP, 1 mM pH 7.6 Tris-HCl, 0.1 mM EDTA, 1U TdT enzyme (Promega)). For nucleus visualization, the slides were counter-stained utilizing DAPI. Using a Nikon TS2 fluorescent microscopy, the fluorescent green apoptotic cells were examined and imaged.

Fly brains were dissected in cold PBS and fixed for 45 min in 4% PFA at RT. After three washes of 10 min in PBS, the brains were incubated 10 min in 50 μl of proteinase K (20 μg/ml in PBS) and then washed twice for 10 min in PBS (to stop the permeabilization). For the positive control, a 10 min incubation in DNase I buffer was followed by a 10 min incubation in DNase I (7 U/ml in DNase I buffer) and then washed three times in double-distilled water (ddH₂O). The brains for all the conditions were then immersed for 30 min in equilibration buffer and incubated 1 h at 37°C with a solution containing 44 μl of equilibration buffer, 5 μl of marked nucleotide mixture, and 1 μl of TdT enzyme (Promega). The reaction was stopped for 15 min in 2× SSC solution (diluted in ddH₂O) and washed three times in PBS. The brains were then mounted in Vectashield (Vector Laboratories).

TUNEL assay was performed by automated staining using Bond RX (Leica Biosystems) immunostainer. Paraffin slides were dewaxed in Bond dewax solution (product code AR9222, Leica Biosystems). Antigen retrieval was performed with Protease incubation for 25 min at 37°C. TdT Enzyme incubation was performed for 20 min with antibody diluent (Leica AR9352), TdT Buffer (Promega M1893), DIG-11-dUTP (Roche, Ref 11570013910) and TdT Enzyme (Promega M1875) followed by incubation with mouse anti DIG-FITC (1:500) for 15 min and Rabbit anti FITC (1:1000) for 15 min. The reaction was visualized using AP (Alkaline phosphatase)-rabbit polymer for 15 min and fast red as red chromogen (Red polymer refine Detection, Leica Biosystems, Ref DS9390). Finally, the samples were counterstained with Haematoxylin and mounted with Aquatex (Merck). Stainings were performed by the translational research unit of the institute of Pathology, University of Bern.