Sigmafast tablet

Blood samples were taken on days 1, 14, and 42 immediately before infliximab infusion and on follow-up visits, each one week after infusion (Figure 1). On the first blood draw for serum, immediately before infusion 1 (Inf1), CD patients were naïve to infliximab. This time point was used as a baseline to normalize data. To verify a drug (i.e., infliximab) effect in relation to circulating I-FABP, TNFα and CRP levels were measured.
A 50X protease inhibitor cocktail solution was prepared by dissolving a SigmaFast tablet (catalog number S-8830, Sigma-Aldrich, USA) in 2.2 ml deionized H₂O and adding 5.5 μl 10 mM KR-62436 (catalog number K4264, Sigma-Aldrich) in DMSO along with a separate 68 mM 10X EDTA stock [13 (link)]. After vortexing, the 50X cocktail was pipetted immediately into blood tubes to 1X final concentration. Because this cocktail was not added at the time of blood draw in the case of the biobanked infliximab infusion and follow-up samples, it was added along with final 1X EDTA (for comparisons to plasma) prior to thawing in order to minimize degradation during or subsequent to thawing and to permit identical chemical composition as with all the recently obtained samples (i.e., controls) they were compared against, into which this cocktail was added at the time of blood draw.

Venous blood was collected by trained nurses 15 minutes before breakfast and at 15, 35, 65, 95, 125, 185 and 230 minutes after breakfast. Blood samples were collected into ice cold tubes prepared with either potassium EDTA or lithium heparin (Sarstedt, Nümbrecht, Germany). Immediately after blood sampling 160 μl protease inhibitor cocktail (1 SigmaFast tablet (Sigma S8820) in 2.2 ml H2O + 5.5 μl 10 mM KR-62436(DMSO)) were added to the test tube to prevent breakdown of appetite related gut peptides and incretines, which may be analyzed later for other purposes. The samples were kept on ice and processed immediately. Blood plasma was separated from buffy coat and erythrocytes by centrifugation at 2500 g for 10 min (4°C), aliquoted into 2.0 mL screw cap micro tubes (Sarstedt, Nümbrecht, Germany) and immediately placed in a freezer at -20°C. Plasma samples were transferred and stored in a freezer maintaining a temperature of -80°C. Analyses of serum insulin and plasma glucose concentrations were performed by established routine methods at the certified laboratory of the Department of Clinical Chemistry at Uppsala University Hospital. Serum insulin was analyzed on a Roche cobas 8000 e602 module (Roche Diagnostics GmbH, Mannheim, Germany) and plasma glucose on an Architect c8000 and c16000 (Abbott, Abbott Park, Illinois, USA).

Whole‐mount in situ hybridization was performed based on the protocol described in Pearson et al. (2009) but with the following modifications. For fixation, 7.5% N‐acetyl cysteine was used, the reduction solution step was omitted, and worms were stored in MeOH. For bleaching, samples were rehydrated into Phosphate Buffered Saline with Triton X (PBSTx), rinsed in 1× saline sodium citrate (SSC), then placed in formamide bleaching solution overnight as described in King & Newmark (2013). Prior to post‐fix, samples were rinsed in 1× SSC, then twice in PBSTx, followed by a 10 μg/mL proteinase K treatment. For NBT (nitro blue tetrazolium)/BCIP (5‐bromo‐4‐chloro‐3‐indolyl‐phosphate) developing, Sigma FAST tablet (B‐5655) was dissolved in 7.3 mL of alkaline phosphatase (AP) buffer with 10% polyvinyl alcohol plus 2.7 mL nuclease‐free water. Smed‐tyrosinase (Lapan & Reddien 2011) probe was used at 1 ng/μL. Anti‐digoxigenin‐AP (Roche, Basel, Switzerland) was used, 1:3000.