Mouse anti rat cd68 clone ed1

Spleens and livers were weighed and then fixed in 10% buffered formalin and processed into paraffin by the Histopathology Department at the Royal (Dick) School of Veterinary Studies using standard procedures. Slides were stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed with mouse anti-rat CD68 (Clone ED1, 1:500, Bio-Rad). For Oil red O staining, formalin-fixed livers were placed in 18% sucrose at 4°C overnight and cryosections prepared as described in (45 (link)). Staining was performed as described in Ref. 29 (link). Sections were imaged using a Nanozoomer digital scanner and viewed using NDP.view 2 (Hamamatsu, Japan). Oil red O staining was imaged with standard light microscopy using ZEN software (Zeiss).

For immunofluorescence staining, rat heart specimens were embedded in Tissue-TeK medium, frozen on dry ice, and stored at −80°C. 10-μm-thick cross-sections were prepared and parallel sections were immunostained. Briefly, slides were thawed at room temperature and fixed with acetone, rehydrated in phosphate buffer saline, blocked with 10% donkey serum, and incubated 3 h with primary antibodies diluted with 2.5% bovine serum albumin (BSA). Primary antibodies include mouse anti-rat CD68 (clone ED-1, Bio-Rad) for macrophages and rabbit anti-MR antibody (polyclone, Abcam) for MR. Corresponding secondary antibodies were conjugated with Alexa 488 and Cy5. DAPI was used for DNA staining. For negative controls, staining was performed without primary antibodies. Stained sections were analyzed using a DM6000 fluorescence microscope and a SP8 confocal laser scanning microscope (Leica, Mannheim, Germany). Fluorophores were visualized by using a 488 nm excitation filter and 505/530 nm emission filter for Alexa 488, and a 633 nm excitation, and 650 nm emission long-pass filter for Cy5. For 3D imaging, z-stacks were prepared at 1 μm intervals with a scan zoom factor 2 using 100× objective and then processed LasX software (Leica).