Donkey anti rabbit igg488

Following bioreactor perfusion, kidneys were fixed in 10% neutral buffered formalin (NBF), imbedded in paraffin wax, and sectioned for immunochemistry (IHC). Primary antibodies for IHC were LTL (Vector labs, B1325, Newark, CA, USA), E cadherin (R&D, AF748, Santa Clara, CA, USA), Nephrin (R&D System, AF4269, Toronto, ON, Canada), KSP (Novus Biologicals, NBP1-59248, Littleton, CO, USA), AQP2 (Alomone Labs, AQP-002, Jerusalem, Israel). The primary antibodies were diluted in 0.1%FBS/0.1%Triton/PBS at a 1:100 ratio (except for E cadherin which was diluted at a 1:50 ratio). The secondary antibodies used were: Strepavidin 488 (Invitrogen, S32354, Waltham, MA, USA), Donkey anti-goat IgG Cy5 (Abcam, ab6566, Cambridge, UK), Donkey anti-sheep IgG NL557 conjugated antibody (R&D Systems, NL010), Donkey anti-rabbit IgG594 (Invitrogen, A21207), Donkey anti-rabbit IgG488 (Invitrogen, A21206). They were diluted in the same solution as the primary antibodies at a 1:500 ratio. Cell nuclei were stained with DAPI. Hematoxylin and Eosin (H&E) staining was also done to visualize tissue morphology.
Kidney was also stained with Periodic-acid Schiff (PAS) reagent staining. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was done as previously described [12 (link)].

Cells were dissociated with trypsin for 5 min, neutralized using a medium, and gently centrifuged (1000× g, 10 min). The supernatants were removed and the cells were resuspended in DPBS with 2% normal goat serum at RT for 30 min. Then, the cells were cultured with SSEA1 (1:100, Santa Cruz, Dallas, TX, USA) solution for 1 h at RT, washed 3 times (1000× g, 5 min) with DPBS, and incubated with the secondary antibody (donkey anti-rabbit IgG-488, 1:500, Invitrogen, Waltham, MA, USA) for 20 min at RT in the dark. Afterward, cells were washed 3 times (1000× g, 5 min) and resuspended in DPBS in a Flow cytometer (Cytomics FC 500, Beckman Coulter, Brea, CA, USA). The data were analyzed using FlowJo flow cytometry software (version 7.6.1, Tree Star Inc., Ashland, OR, USA).