Goat anti hnf4α

Whole-mount immunohistochemistry was performed using a modification of
the protocol from Ahnfelt-Rønne et al.
(2007) as previously described (Havrilak and Shannon, 2015b (link)). Optical sections were captured on a
Nikon AiRsi inverted laser microscope, and 3D images were created by a composite
of z-stacks using Bitplane Imaris software to process the fluorescent images. To
calculate the volume of tissue stained for ALB, we created isosurfaces using
smoothing thresholds and intensity values that allowed filtering out antibody
aggregates or any non-specific staining.
The primary antibodies used were: rabbit anti-NKX2-1 (1:3000, Seven
Hills Bioreagents), guinea pig anti-NKX2-1 (1:500, Seven Hills Bioreagents),
goat anti-endomucin (1:500, R & D Systems), rat anti-E-cadherin (1:2000,
R & D Systems), rabbit anti-PAX8 (1:500, Protein Tech), rat anti-PECAM-1
(1:500, BD Pharmigen), goat anti-HNF4α (1:200, Santa Cruz), goat
anti-PDX1 (1:5000, Abcam), goat anti-ALB (1:1000, Bethyl Laboratories), goat
anti-FOXA2 (1:500, Santa Cruz), and rat anti-LIV2 (1:200, MBL). Secondary
antibodies used were: Alexa Fluor 647 donkey anti-rabbit IgG, Alexa Fluor 568
anti-goat IgG, Alexa Fluor 488 donkey anti-rat IgG, Alexa Fluor 488 donkey
antimouse IgG (1:500, all from Life Technologies).

For immunostaining of embryonic tissue, pregnant females were anesthetized and the embryos collected at E12, E15 and E18. The embryos were then fixed in a solution of 4% paraformaldehyde. For immunostaining of adult tissue, mice were transcardially perfused with 4% paraformaldehyde. Tissue sections were processed for immunofluorescence using standard procedures. The primary antibodies used for immunohistochemistry were as follows: rabbit anti-vesicular acetylcholine transporter (VAChT, 1:500, Synaptic Systems), mouse anti-synaptophysin1 (1:500, Synaptic Systems), rabbit anti-tyrosine hydroxylase (TH, 1:2000, Immunostar), goat anti-HNF4α (1:500, Santa Cruz), and guinea-pig anti-insulin (1:500, Abcam). For detailed information regarding image acquisition and quantification, see the Supplemental Experimental Procedures.