Casein solution

Lymph nodes were embedded in TissueTek OCT compound and 8 µm cryostat sections were prepared and fixed in acetone. After peroxidase inactivation and blocking of unspecific binding sites with Casein Solution (Vector Laboratories) supplemented with mAb 2.4G2 (100 µg/ml), sections were stained with FITC-coupled OX-7 (anti-Thy-1.1), Alexa Fluor 555–coupled RA3-6B2 (anti-B220), and Alexa Fluor 594–coupled GK1.5 (anti-CD4). Signals for Thy-1.1 were amplified with peroxidase-conjugated anti-FITC followed by Alexa Fluor 647–coupled tyramide (Invitrogen). Nuclei were stained with DAPI. Whole lymph node sections were captured on an LSM 780 with ZEN2010 imaging software using the tile-scan function (Carl Zeiss).
At least 6 sections from different areas of each lymph node were used for quantification of signals. For this purpose, total lymph node area (any signal) and B cell follicles (B220⁺) were defined and transgenic T cell signal (Thy-1.1) was separated from background and unified by setting an appropriate threshold. Localization of T cells was determined by automatically counting pixels positive for the Thy-1.1 signal in the distinct lymph node compartments. Mean B cell follicle sizes were similar between groups.

PBS (7.0–7.5 μl) were mixed in a microtube with 0.5–1.0 μl primary antibody and 1 μl QDs, and vortexed for 5 min at low speed (total volume 9 μl). Subsequently, 1 μl of casein (10 × Casein Solution, Vector Laboratories Inc; Burlingame, CA, 94010) was added to the tube and vortexed for an additional 5 min. A 35-mm Petri dish was covered with parafilm and a drop of 100 μl conditioned medium was added. The antibody-coupled QDs were added at a dilution of 1:1,000 (so 1 μl of the above-prepared solution) and mixed. Then, using a scalpel a slice was cut out from the membrane and transferred to the drop in the Petri dish. The Petri dish was returned to the incubator for 5–10 min. After, the slice was washed three times in HEPES-buffered extracelluar solution containing 0.5-1% BSA (albumin from bovine serum; Sigma A7638 10 G). For imaging, slices were transferred in HEPES-buffered extracellular solution (containing in mM: 145 NaCl, 2.5 KCl, 2 MgCl₂, 2 CaCl₂, 10 HEPES and 10 D-glucose, pH 7.4).

Hearts were harvested and fixed in 5% paraformaldehyde. The left ventricle was cut into six transversal, 1 cm-thick slices from apex to base. Then, from each heart, several approximately 1 cm × 1 cm × 1 cm large tissue samples were collected, representing the left ventricular border zone of MI, the core region of MI, and regions of viable myocardium. Specimens were paraffin-embedded and cut into 5 µm-thick tissue sections that were mounted on glass slides and stained with Masson’s Trichrome staining, or processed with fluorescence immunohistochemistry.
Fluorescence immunohistochemistry was performed on de-paraffinized and rehydrated sections that were washed with PBS containing 0.3% Triton X-100 (Sigma Aldrich, St. Louis, MO, United States) and blocked with 10% casein solution (Vector Laboratories, Burlingame, CA, United States) for 30 min at room temperature. Then, sections were incubated overnight with diluted Rabbit anti-von Willebrand factor (vWF) primary antibody (Abcam) or diluted Rabbit anti-adiponectin (Cy5 conjugated) primary antibody (Biorbyt, San Francisco, CA, United States), and subsequently for 1 h with diluted Goat anti rabbit-IgG secondary antibody (Cy5 conjugated) (Thermo Scientific, Waltham, MA, United States). Counterstaining of nuclei and mounting were performed with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories).