Alex594 conjugated goat anti rabbit igg

iPSCs were plated at 2 × 10³ onto four well plates in 2i/LIF. After 24 hours, the cells were washed with PBS, fixed in 4% paraformaldehyde (PFA), blocked and permeabilized with 1% bovine serum albumin and 3% serum in PBS with 0.3% Triton X‐100. The specimens were incubated with anti‐SSEA‐1 (BD Biosciences) or anti‐Nanog (Abcam, Cambridge, UK, http://www.abcam.com) antibodies at 4°C overnight. They were then rinsed and incubated with Alex594‐conjugated goat anti‐Mouse IgM and Alex594‐conjugated goat anti‐Rabbit IgG (Invitrogen) antibodies and counterstained with DAPI.

Middle parts of bladder tissues were fixed in 4% paraformaldehyde in PBS at 4°C overnight, after which the tissue was transferred to 30% sucrose in PBS at 4°C overnight. The tissues were embedded in Optimal Cutting Temperature (OCT) and stored at −80°C.
For immunofluorescence staining, sections were incubated with 3% goat serum for 30 min at room temperature. Bladder sections were then incubated with primary antibodies overnight at 4°C, followed by Alex594-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA, 1 : 200) for 1 h at room temperature. The primary antibodies used in the present study was rabbit anti-CD31 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 : 200). EdU staining was performed as described before.
For Masson's trichrome staining, the slides were stained using commercial available kits (Jiancheng, Nanjing, China) following the protocols provided by the manufacturer.