Rabbit anti hes5

The protein expressions of Hes1 and Hes5 in PBMCs were measured as previously described [20 (link)]. Briefly, total proteins, which were extracted from DMSO or DAPT-treated PBMCs, were loaded on SDS-PAGE gel, and were electroblotted onto PVDF membrane. The membrane was soaked in 5% non-fat milk containing 0.05% Tween 20 in PBS for 2 h, and was then incubated overnight in the presence of rabbit anti-Hes1 (Abcam, Cambridge, MA, U.S.A.; 1:1000 dilution), rabbit anti-Hes5 (Abcam; 1:1000 dilution), or mouse anti-GAPDH (Abcam; 1:2000 dilution). Horseradish peroxidase-conjuated goat anti-rabbit or goat anti-mouse antibody IgG (Abcam; 1: 2000 dilution) was then added for additional 2-h incubation. Antigen–antibody complexes were observed by enhanced chemiluminescence (Western Blotting Luminol Reagent, Santa Cruz, CA, U.S.A.).

Mouse anti-CNPase, rabbit anti-Hes1, and rabbit anti-Hes5 antibodies were purchased from Abcam Corporation (Cambridge, UK). Anti-MBP antibodys were obtained from Millipore Corporation (Billerica, MA) for immune staining or ABcam (ab62631) for Western blot, and immobilon Western chemiluminescent HRP substrate were obtained from Millipore Corporation. Mouse anti-β-actin monoclonal antibody was purchased from CWBIO Corporation (Beijing, China). HRP-linked anti-rabbit and anti-mouse IgG antibodies were from Cell Signaling Corporation (Beverly, MA). Alexa Fluor 594 Donkey anti-rat IgG was from Invitrogen Corporation (San Diego, CA), and primers of Notch1, Notch2, Notch4, Hes1, and Hes5 were synthesized by TaKaRa Corporation (Seta, Japan). PrimeScript RT reagent Kit with gDNA Eraser, SYBR Premix Ex Taq (TliRNaseH Plus), and EASY Dilution were also from TaKaRa Corporation. Cuprizone (bis-cyclohexanoneoxalydihydrazone, CPZ) was purchased from Sigma Corporation (Ronkonkoma, NY). Quetiapine was a generous gift from Professor Li Xinmin, University of Manitoba, Canada. Halothane was obtained from Halocarbon Laboratories (Kinderkamack, NY). Biodentine dental cement was obtained from Septodont Corporation (Saint Maur des Fossés, France). DMSO was obtained from Sigma Corporation, and MW167, a γ-secretase II inhibitor, was purchased from Calbiochem Corporation (La Jolla, CA).

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).