Low light time lapse microscope

Embryos still in decidua were fixed in 4% Paraformaldehyde (PFA) for two hours, before they were soaked in 30% sucrose and mounted in OCT compound. 7 µm sections were prepared using a cryostat. The sections were streptavidin/biotin blocked followed by serum blocking (PBS with 10% FCS, 0.05% Tween20 and of 10% goat serum (DAKO) for 1 hour before the sections were incubated with primary antibodies at 4 °C overnight in blocking buffer. Primary antibodies used were rabbit anti –GFP (598, polyclonal, MBL) (1/200); rat anti-mouse c-kit (553352, clone 2B8, BD Biosciences) (1/100); biotinylated rat anti-mouse Tie2 (13–5987, clone TEK4 eBioscience) (1/100). After staining, the sections were washed three times in PBST for 15 minutes each and then incubated with fluorochrome-conjugated secondary antibody at room temperature for 1 hour. These were Alexa Fluor® 488 Donkey Anti-Rabbit IgG (H + L) (A21206, Life Technologies); Alexa Fluor® 647 Goat Anti-Rat IgG (H + L) (A21247, Life Technologies); Streptavidin, Alexa Fluor® 555 Conjugate (S32355, Life Technologies). All secondary antibodies were used at 1/400 dilution. The sections were then further washed three times in PBS and mounted using Prolong Gold anti-fade medium with DAPI (Life Technologies). Images were taken using a low-light time lapse microscope (Leica) using the Metamorph imaging software and processed using ImageJ.

AGM sections were stained as described previously (Thambyrajah et al., 2016a (link)) and mounted using Prolong Gold Antifade medium with DAPI (Life Technologies). Images were taken using a low-light time-lapse microscope (Leica) using the Metamorph imaging software, and images were processed using ImageJ.

Dissected fetal livers were fixed in 4% Paraformaldehyde (PFA) overnight, before they were soaked in 30% sucrose and mounted in OCT compound. 10μm sections were prepared using a cryostat. The sections were incubated in blocking buffer (PBS with 10% FBS, 0.05% Tween20 and 10% goat serum (DAKO)) for 1 hour before the sections were incubated with primary antibodies at 4°C overnight in blocking buffer.
Primary antibodies used in this study were rabbit anti-GFP (598, polyclonal, MBL) (1/200); and purified rat anti-mouse CD31 (553370, MEC13.3, BD Biosciences) (1/100).
Sections were washed three times in PBST (PBS with 0.05% Tween20) for 15 minutes each and then incubated with fluorochrome-conjugated secondary antibody at room temperature for 1 hour.
Secondary antibodies used in this study include Alexa Fluor 488 Goat Anti-Rat IgG (A11006, Life Technologies); and Alexa Fluor 647 F(ab')₂ Fragment of Goat Anti-Rabbit IgG (H+L) (A21246, Life Technologies). All secondary antibodies were used at 1/400 dilution.
Sections were further washed three times in PBS and mounted using Prolong Gold anti-fade medium with DAPI (Life Technologies). Images (of Alexa Fluor 488, Alexa Fluor 647, DAPI and endogenous RFP) were taken using a low-light time lapse microscope (Leica) using the Metamorph imaging software and processed using ImageJ.