Whole-mount immunohistochemistry was performed using a modification of
the protocol fromAhnfelt-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).
the protocol from
(2007)
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).