Placentas at 18.5 dpc were collected and fixed overnight in 4% PFA at 4°C. The tissues were then processed for paraffin embedding. Sections of size 5 μm thickness were sliced,
deparaffinized, and rehydrated. Antigen retrieval was performed by boiling the sections for 20 min in a citrate-based antigen-unmasking solution. Non-specific binding was blocked using 4%
normal horse serum for 30 min.
The following primary antibodies were used: Rabbit anti-Nrk (Merck, HPA017238, 1:100), goat anti-endomucin (R&D Systems, MN, USA, AF4666, 1:200), goat anti-proliferin (R&D Systems,
AF1623, 1:200), and goat anti-IGF1r (R&D Systems, AF305-NA, 1:200), which were incubated overnight at 4°C. The secondary antibodies used were Alexa Fluor® 488 donkey anti-rabbit IgG
H&L (Jackson ImmunoResearch, 711-545-152, 1:500) and Alexa Fluor® 594 donkey anti-goat IgG H&L (Abcam, Cambridge, UK, ab150132, 1:500), which were incubated for 1 h at 20–25°C.
Counterstaining and mounting were performed using VECTASHIELD® with DAPI (Vector Laboratories, H-1200).
deparaffinized, and rehydrated. Antigen retrieval was performed by boiling the sections for 20 min in a citrate-based antigen-unmasking solution. Non-specific binding was blocked using 4%
normal horse serum for 30 min.
The following primary antibodies were used: Rabbit anti-Nrk (Merck, HPA017238, 1:100), goat anti-endomucin (R&D Systems, MN, USA, AF4666, 1:200), goat anti-proliferin (R&D Systems,
AF1623, 1:200), and goat anti-IGF1r (R&D Systems, AF305-NA, 1:200), which were incubated overnight at 4°C. The secondary antibodies used were Alexa Fluor® 488 donkey anti-rabbit IgG
H&L (Jackson ImmunoResearch, 711-545-152, 1:500) and Alexa Fluor® 594 donkey anti-goat IgG H&L (Abcam, Cambridge, UK, ab150132, 1:500), which were incubated for 1 h at 20–25°C.
Counterstaining and mounting were performed using VECTASHIELD® with DAPI (Vector Laboratories, H-1200).