Ab196990

For immunohistochemistry staining, the skin and large intestine sections were treated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity and incubated with blocking buffer containing 2% bovine serum albumin. Then, the sections were incubated with the primary antibodies, followed by incubation with the biotinylated secondary antibodies. The following primary antibodies were used: anti-Filaggrin (1:100 dilution, #ENZ-ABS181, ENZO, USA), anti-TSLP (1:100 dilution, #ab196990, Abcam, USA), anti-ZO-1 (1:200 dilution, #61-7300, Invitrogen, USA), anti-IL-4 (1:100 dilution, #SC-53084, Santa Cruz Biotechnology, USA), and anti-Claudin-1 (1:100 dilution, #SC-166338, Santa Cruz Biotechnology, USA). Lastly, the color was revealed using 3,3′-diaminobenzidine (DAB). For counting staining, tissue sections were stained with H&E. The observation and analysis of sample images were conducted using a Nikon Eclipse Ni (Nikon Corporation, Japan). For the quantitative analysis, images of three samples from each group were analyzed using the Nikon NIS-Elements image software.

Mice were housed (n = 1 or 2) in metabolic cages, and 48 hr collections (1–2 mice per urine pool) were made under mineral oil into vessels containing crystal thymol as a preservative. Urinary oxalate (mg/dl) and creatinine (mg/dl) concentrations were determined in acidified (HCl) samples collected from all mice over a 48 hr period by Litholink Corp (Chicago, IL). Fecal pellets were collected at the end of the urine collections. Fecal pellets were acidified using 2M HCl, vortexed for 20 min, and then centrifuged at 21,000 g at room temperature, and supernatant fecal water collected using described methods (Jiang et al., 2011 (link)). Fecal water oxalate was measured using an oxalate calorimetric assay (Abcam, ab196990, Cambridge, UK) per the manufacturer’s instructions. Technical replicates are repeated measurements of the same sample that represent independent measures of the random noise associated with protocols or equipment. Fecal oxalate and qPCR were measured in duplicates.