Goat anti pdx1

Pancreas sections were prepared from MT-tg (n = 5) and WT male mice (n = 5) at 8 weeks of age. At least three slides from each mouse were examined. Immunostaining was conducted using routine procedures for paraffin embedding and sectioning as described previously [32 (link)]. Briefly, sections were deparaffinized, rehydrated, pretreated with target antigen retrieval solution, washed with PBS, blocked with serum, and incubated with primary antibodies [guinea pig anti-insulin (Abcam, Cambridge, MA, USA), diluted to a ratio of 1:50; mouse anti-horse MT (Dako, Carpinteria, CA, USA), diluted to 1:50, or rabbit anti-glucagon (Abcam), diluted to 1:50; goat anti-PDX1, diluted to 1:50 (Abcam)] at 4°C overnight. After incubation, immune complexes were detected by double-labeling immunohistochemistry, incubation with fluorescein isothiocyanate (FITC)-labeled anti-guinea pig IgG, and Cy3-conjugated anti-mouse IgG or Cy5-conjugated anti-rabbit IgG and Cy3-conjugated anti-goat IgG (1:200; all from Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Fixed and cryosectioned samples were blocked with 3% (w/v) BSA in PBS and permeabilized with 0.3% v/v Triton X-100 for 1 h at room temperature. Samples were incubated with the following primary antibodies in 1% w/v BSA for 1 h at room temperature: mouse anti-osteocalcin (1:200; cat#, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-collagen type II (1:200; II-II6B3-C, DSHB), goat anti-PDX-1 (1:200; 47,383, Abcam, Cambridge, UK), guinea pig anti-insulin (1:200; ab7842, Abcam, Cambridge, UK), and rabbit anti-connexin 36 (1:200; 364,600, Invitrogen, Carlsbad, CA, USA). Samples were washed with PBS and incubated with the following secondary antibodies: anti-rabbit Alexa 555 (1:200; A21428, Thermo Fisher Scientific, Waltham, MA, USA), anti-rabbit Alexa 647 (1:200; cat#, Thermo Fisher Scientific, Waltham, MA, USA), anti-mouse Alexa 488 (1:200; A11001, Thermo Fisher Scientific, Waltham, MA, USA), anti-guinea pig Alexa 488 (1:200; A11073, Thermo Fisher Scientific, Waltham, MA, USA), and anti-goat Alexa 555 (1:200; A31573, Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with 2 mg/mL Hoechst 33,342 (1:1000; H21492; Molecular Probes, Eugene, CA, USA) for 10 min at room temperature. Fluorescence images were acquired using a confocal microscope (LSM 710; Carl Zeiss, Oberkochen, Germany) at the Soonchunhyang Biomedical Research Core Facility of the KBSI.

Differentiated cells at specific stages were fixed with 70% ethanol and blocked overnight with 6% BSA in PBST. Cells were stained with the primary antibodies goat anti-PDX1 (1:100; Abcam), goat anti-SOX9 (1:50; R&D Systems), mouse anti-NKX6.1 (1:100; DSHB), goat anti-NKX6.1 (1:50; R&D Systems), and mouse anti-Ki67-647 conjugate (1:50; BD Biosciences) for 3–4 h at room temperature. The cells were incubated with Alexa-fluor secondary antibodies (1:200; Molecular Probes, ThermoFisher Scientific) for 40 min at room temperature. The results were analyzed using the BD Accuri C6 flow analyzer and the results were processed using FlowJo.