Nis elements br 4.13.00 64 bit image analysis system

The heart tissues were fixed in 10% neutral buffered formalin after excision and weighing and processed using standard techniques. Five μm histological sections were prepared and stained with hematoxylin-eosin (H&E) and Masson’s Trichrome stains. Immunohistochemical staining was performed using antibodies for F4/80 (1:200, Abd serotec Raleigh, NC), collagen III (1:20, SouthernBiotech, Birmingham, AL), alpha SMA (1:500, Abcam, Cambridge, MA), iNOS (1:800, Abcam, Cambridge, MA) and CD206 (1:800, Abcam, Cambridge, MA). All measurements and quantifications were performed in a random blinded fashion using Olympus Bx50 microscope (Olympus Optical Co. Ltd., Buffalo Grove, IL), Micropublisher 3.3 RTV camera (QImaging, Surrey, BC, Canada). The amount of necrosis in each heart section was assessed on H&E sections. Quantitative analysis of % area of fibrosis for trichrome and % positively stained area for F4/80, alpha SMA and collagen III was performed using NIS elements BR 4.13.00 64-bit image analysis system (Nikon Instruments INC., Melville, NY) at 200X magnification. The number of iNOS⁺ cells was counted at 200X and CD206⁺ cells at 400X high field magnification.

Histologic and immunohistochemical analysis was performed on the subset of animals for which imaging studies were performed (see above). The kidney tissues were fixed in 10% neutral buffered formalin and processed using standard techniques. 5 µm histological sections were prepared and stained with hematoxylin-eosin (H&E). Immunohistochemical staining was done for anti-F4/80 (1:200, Abd Serotec Raleigh, NC) and anti-CD3 (1:100, Dako Agilent, Santa Clara, CA). Sections were stained with Sirius red to quantitate matrix deposition. All measurements and quantifications were performed in a blinded fashion using Olympus Bx50 microscope (Olympus Optical Co. Ltd., Buffalo Grove, IL, USA), Micropublisher 3.3 RTV camera (QImaging, Surrey, BC, Canada). The area of atrophy was calculated on H&E sections as percentage of atrophic tubules over the cortical area, as previously described^{9 (link),20 (link)}.
Quantitative analysis for F4/80 CD3, and Sirius red was done by calculating % positive stained area for F4/80, CD3, and Sirius red using NIS elements BR 4.13.00 64-bit image analysis system (Nikon Instruments INC., Melville, NY) at 200X magnification.

Histologic and immunohistochemical analysis was performed on right kidney tissue from mice that underwent UUO or Sham surgery. The kidney tissues were fixed in 10% neutral buffered formalin and processed using standard techniques. 5 μm histological sections were prepared and stained with hematoxylin-eosin (H&E). Renal atrophy was semi quantitatively assessed as the percentage of cortical surface area occupied by atrophic tubules, compared to the entire cortical surface area, according to methods previously established in our laboratory [14 (link), 15 (link)]. Immunohistochemical staining was done for anti-F4/80 (1:200, BIO-RAD, Cat. No. MCA497RT), anti-KLF11 (1:1600, Novus Biological, Cat# H00008462-M03), anti-CD3 (1:100, Agilent Dako, Code Number A045), anti-CD68 (1:200, Abcam, Cat# ab125212), anti-CD163 (1:400, Abcam, Cat# ab182422), and anti-CD206 (1:800, Abcam, Cat# ab64693). Sections were stained with Sirius Red to quantitate matrix deposition. Ten random fields per kidney section were examined for each marker (F4/80, CD3, CD68, CD163, CD206 and Sirius Red) and the average of positive areas was expressed as percentage of total analyzed area. All measurements and quantifications were performed in a blinded fashion using NIS elements BR 4.13.00 64-bit image analysis system (Nikon Instruments INC., Melville, NY) at 200 X magnification.