Eclipse ni microscope

Fluorescent microscopy images were obtained at the same laser intensities and exposure times using either a Nikon Eclipse Ni microscope (×20 objective) or a Zeiss Axio Observer Z1 inverted microscope (×10 and ×20 objective). Images obtained with the latter were further processed on the Zeiss Zen 3.4 (blue edition) software to improve their clarity via the “Deblurring” function.

Frozen histological sections were immersed in pre-cooled methanol (−20 °C) for 15 min. Blocking was performed for 1 h at room temperature with a solution composed of 1X TBS, 10% BSA and 0.3% Triton-X. Sections were incubated overnight (4 °C) with primary antibodies against CD-3 (dilution 1:50, DAKO A0452), CD-20 (dilution 1:200, DAKO M0755), granulysin (dilution 1:100, Santa Cruz sc-271119), and PNAD (dilution 1:200, BD Biosciences 553863). AlexaFluor® 488 (dilution 1:200, Invitrogen A21121 and A21212), AlexaFluor® 546 (dilution 1:200, Invitrogen A11030), and AlexaFluor® 594 (dilution 1:200, Invitrogen A11037) secondary fluorescent antibodies were applied for 1 h at room temperature. Nuclei were labeled with DAPI (dilution 1:1000, Invitrogen D1306) and slides were mounted with Fluoromount-G® (SouthernBiotech). Stained slides were evaluated using a Nikon Eclipse NI microscope and a Zeiss Axio Observer 7 with appropriate filters.

Immunostaining was performed on paraffin-embedded sections (4 µm) to determine the distribution of mouse ACE and human AGT in the kidney. Deparaffinized sections were incubated with an antigen retrieval reagent (Cat # HK547-XAK; BioGenex) for 20 min at 90°C. Non-specific binding sites were blocked using goat serum for 20 min at room temperature. Sections were then incubated with rabbit anti-mouse ACE antibody (Cat # ab254222; abcam) or anti-human AGT antibody (Cat # ab276132; abcam) diluted in primary antibody diluent (Cat #: GTX28208; GeneTex) for 12 h at 4°C. Goat anti-rabbit IgG conjugated with horseradish peroxidase (Cat # MP-7451; Vector laboratories) was used as the secondary antibody. ImmPACT® AEC (Cat # SK4205; Vector) was used as the chromogen, and hematoxylin (Cat # 26043–05; Electron Microscopy Sciences) was used for counterstaining. Histological images were captured using either an Eclipse Ni microscope or Zeiss Axioscan 1 or 7.