Rabbit anti hes1

Kaiso^Tg intestinal tissues were formalin-fixed and paraffin embedded as previously described [19 (link)]. Periodic acid-Schiff (PAS) staining was performed by the John Mayberry Histology Facility at McMaster University. Immunohistochemistry (IHC) analysis of all other protein targets was performed as previously described [19 (link)], with the following modifications: antigen retrieval for chromogranin A was accomplished by heating tissues in 10 mM sodium citrate, pH 6.0 for 10 min at sub-boiling temperature; retrieval for NICD, Dll-1, Dll-4, Hes1, and Hes5 was accomplished by heating tissues at sub-boiling temperature for 15 min in TE-Tween (Tris EDTA, 0.05% Tween), pH 9.0; and retrieval for lysozyme was performed with 200 μg/mL proteinase K, 50 mM Tris pH 7.4 at RT for 5 min. Tissues were incubated with the following primary antibodies overnight at 4 °C at the indicated dilutions: rabbit anti-lysozyme (Thermo Scientific cat. #PA1–29680; 1:50); rabbit anti-chromogranin A (Abcam cat. #ab15160; 1:500); rabbit anti-Cleaved Notch 1 Val-1744 (Cell Signaling Technology cat. #4147; 1:75); rabbit anti-Hes1 (Cell Signaling Technology cat. #11988S; 1:80); rabbit anti-Hes5 (Abcam cat. #ab65077; 1:125); rabbit anti-Dll-1 (Abcam cat. #ab84620; 1:100); and goat anti-Dll-4 (R&D Systems cat. #AF1389).

For histology, tissues were fixed in 10% neutral buffered formalin or Carnoy’s fixative (60% methanol, 30% chloroform, and 10% acetic acid) before paraffin embedding. Images were obtained with an Eclipse i80 microscope (Nikon Instruments, Melville, NY, USA). Paraffin-embedded 5-μm-thick sections were stained with H&E for gross morphology and with Alcian blue to detect goblet cells. Immunohistochemistry and immunocytochemistry were performed with appropriate antibodies on paraffin-embedded sections [18 (link), 21 (link)]. Antibodies used were rabbit anti-DCLK1 (1:200, ab31704) and Anti-CR (1:250, ab37056) from Abcam, Cambridge, UK; mouse anti-Notch1 NICD (1:200, clone OTI3E12) from Origene, Rockville, MD; rabbit anti-Hes1 (1:200, #11988) and mouse anti-Ki-67 (1:200, #9449) from Cell Signaling Technology, Danvers, MA; anti-CD68 (1:200, NB600-985) from Novus Biologicals (Littleton, CO, USA), Anti-Muc2 and anti-NE (1:200) Santa Cruz, Dallas, TX; Anti-CD11c and anti-F4/80 (Thermo Fisher), and anti-Ly6G (R&D, Minneapolis, MN). Antibody controls included omission of the primary antibody or detection of endogenous IgG staining with goat anti-mouse or anti-rabbit IgG (Calbiochem, San Diego, CA, USA).

The deparaffinized and rehydrated coronal sections (4 μm thickness) were prepared as described earlier. Endogenous peroxidase activity was quenched using 3% H₂O₂ for 10 min at RT. Sections were then blocked in 5% bovine serum albumin for 20 min and were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-RBP-Jκ (1:200; 5313T, Cell Signaling Technology) and rabbit anti-Hes-1 (1:200; 11988s, Cell Signaling Technology). Brain slices were then washed with PBS and incubated with biotinylated goat anti-rabbit IgG (1:100; ZSGB-Bio, Beijing, China) for 10 min at RT. Next, brain slices were incubated with horseradish peroxidase–streptavidin reagent for 10 min and developed using diaminbenzidine peroxidase substrate. Images were captured on a light microscope (Leica-DM2500, Wetzlar, Germany).

Whole cell proteins were extracted with RIPA lysis buffer (150 mm NaCl, 50 mm Tris–HCl, 1% Nonidet P-40, and 0.25% sodium deoxycholate) supplemented with 1 mM pervanadate, 1 mM NaF, protease inhibitor cocktail 200X (Sigma-Aldrich), and inhibitor phosphatase cocktail I and II 100X (Sigma-Aldrich). Protein concentrations were determined by Bradford colorimetric assay (Bio-Rad). Total protein extracts (30 μg) were separated by SDS–PAGE, transferered to membranes, and incubated in blocking solution (5% milk and 0.1% Tween-20 in PBS) for 1 h at RT. Membranes were then incubated with primary antibodies overnight at 4°C. The membranes were then washed three times and incubated with anti-mouse or anti-rabbit secondary antibody conjugated with HRP (1:2,500, Cat. No. 1721011, Cat. No. 1706515; Bio-Rad) for 1 h at RT. The blots were visualized with an enhanced chemiluminescent immunoblotting detection system. The antibodies used were as follows: anti-MYHC (1:1,000, Cat. No. MF 20; DSHB), rabbit anti-PPARg (1:1,000, Cat. No. 2443S; Cell Signaling), rabbit anti-HES-1 (1:1,000, Cat. No. 11988S; Cell Signaling), mouse anti-vinculin (1:1,000, Cat. No. ab18058; Abcam), and rabbit anti-actin (1:1,000, Cat. No. A2066; Sigma-Aldrich). Densitometric analysis was performed using ImageJ software.