Biotinylated rabbit anti rat antibody

Serial 6 μm thick cross-sections were made of the aortic sinus area on a cryotome (Reichert HistoStat, Cryostat Microtome). From the first cross-section in which the leaflets of the aortic valves appeared upward, 63 serial cross-sections were obtained, covering the entire aortic sinus area. Of every 3 consecutive cross-sections, they were subjected to anti-CD68 immunohistochemical staining, hematoxylin phloxine saffron (HPS)-staining, and Oil Red O-staining. HPS (HPS, polyscientific) and Oil Red O-staining (Fisher Scientific) were done with standard methods. Anti-CD68-staining was done with a protocol previously reported³⁷. The sections were blocked with rabbit serum (Vectorlabs), incubated for 1 hour with rat anti-mouse CD68 primary antibody (Abdserotec, 1:250 dilution), 10 minutes with biotinylated rabbit anti-rat antibody (Vectorlabs, 1:200 dilution), thereafter 5 minutes with VECTASTAIN ABC-alkaline phosphatase solution (Vectorlabs), and finally for 17 minutes with Vector Red solution (Vectorlabs).
All cross-sections were digitally imaged with a Nikon eclipse E400 microscope, with a 10x eyepiece and 10x lenses, a Nikon DS-U1 camera box and Nikon DS-5M camera. We used NIS-Elements F3.0 software, and imaged at a 1/350s exposure time, 2560 × 1920 pixels, and pixel size of 1.46 μm²/ pix. Software written in Matlab was developed to facilitate automated image analysis.

Histological analysis of PFA-fixed paraffin-embedded tissue sections for
T cell infiltration was performed as previously described [51 (link)]. In brief, 3 μm cross-sections were stained with
a rat anti-humanCD3 antibody (1:200; Serotec, Düsseldorf, Germany)
followed by incubation with a biotinylated rabbit anti-rat antibody (1:200;
Vector Laboratories, Burlingame, CA). Antigen unmasking was achieved by
pre-treating the sections in 1 mM EDTA, pH 8.0 for 30 min in a microwave oven at
850 W. The peroxidase-based ABC detection system (Dako, Hamburg, Germany) and
DAB were used for visualization. Quantification was achieved by taking pictures
with an Olympus BX51 microscope at a 200-fold magnification and counting
individual cells with ImageJ (http://rsb.info.nih.gov/ij/).
Fluorescent immunohistochemical staining was performed using a rabbit
anti-humanCXCL12 antibody (1:200; Peprotech, Hamburg, Germany) and a rat
anti-humanCD3 antibody (1:200) which were developed with a FITC-labeled goat
anti-rabbit (1:750) and a Cy3-labeled goat anti-rat (1:500) secondary antibody
(Dianova, Hamburg, Germany), respectively. DAPI served as a counterstain.
Analysis was performed using a Zeiss fluorescent microscope Axio Observer Z1 and
AxioVision software.