Fitc conjugated donkey anti goat secondary antibody

Paraffin‐embedded kidney sections (4 m) were deparaffinized in xylene and rehydrated in graded alcohol concentrations. Sections were subsequently boiled with 10 mM sodium citrate in a water bath for 30 min. The sections were then blocked in 3% normal donkey serum (diluted in PBS containing 0.1% Triton‐X) for 1 hr at 4°C, and then incubated overnight at 4°C with diluted primary antibodies as follows: goat anti‐NEP (1:500, Cat # AF1126, R&D Systems) and rabbit anti‐ACE2 (1:500, Cat # HPA000288, Sigma). Then, the sections were incubated with CY³ conjugated donkey anti‐goat secondary antibody (1:500, code # 705‐165‐003, Jackson Immunoresearch). For the co‐localization, FITC conjugated donkey anti‐goat secondary antibody (1:500, code # 705‐095‐147, Jackson Immunoresearch) and CY³ conjugated donkey anti‐rabbit secondary antibody (1:500, code # 711‐165‐152, Jackson Immunoresearch) were used and incubated for 2 hr at 4°C. Sections were mounted using a vectashield‐mounting medium (Vector). Images were acquired using a fluorescence microscope (Optronics).
For morphological evaluation of kidney injury, sections were stained with periodic acid‐Schiff (PAS) by AML laboratories. Sections were examined under bright color field and images were acquired using a 40× objective on the Cytation 5 Automated Microscope (BioTek Instruments Inc.).

For pIgR localization lungs were frozen in Tissue-Tek O.C.T. (Sakura Finetek GmbH, Staufen, Germany). 6 µM acetone-fixed cryosections were blocked with anti-CD16/CD32, incubated with goat anti-pIgR primary antibody, blocked with 5% donkey serum and incubated with FITC-conjugated donkey anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA). Alveoli were identified by DAPI nuclear counterstain. Fixed tissue sections were analyzed by fluorescence microscopy, using the inverted Eclipse Ti-U, NIS-Elements imaging software (Nikon, Tokyo, Japan) and ImageJ software.

Rosiglitazone was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Primary polyclonal goat anti‐NEP (Cat # AF1126) and donkey anti‐goat secondary antibody (Cat # HAF017) were purchased from R&D Systems. CY³ conjugated donkey anti‐goat secondary antibody (code # 705‐165‐147), CY³ conjugated donkey anti‐rabbit secondary antibody (code # 711‐165‐152) and FITC conjugated donkey anti‐goat secondary antibody (code # 705‐095‐147) were purchased from Jackson Immunoresearch. Primary polyclonal rabbit anti‐ACE2 form Sigma (Cat # HPA000288). Mouse Albumin ELISA kit was from Bethyl Laboratories (Cat # E90‐134). Mouse Neprilysin DuoSet ELISA development kit from R&D systems (Cat # DY1126). SensoLyte^® 520 NEP activity assay kit from AnaSpec EGT group (Cat# 72223).