Tissue tek octtm

The aortic roots of ldlr^−/− mice fed a high fat, high cholesterol diet for 24 weeks were dissected and embedded in Tissue Tek OCT^TM (Sakura Finetek, Staufen, Germany). Within the aortic root, serial cryostat sections (6 μm, CM3050S, Leica Microsystems, Wetzlar, Germany) at the level of all 3 cusps were prepared. Lesion areas were analyzed for LCN2, endothelial cells (CD31), alpha smooth muscle cells (αSMA), monocytes/macrophages (MOMA-2) as well as the M1 macrophage marker inducible nitric oxide synthase (iNOS) and counterstained with hematoxylin (Carl Roth, Karlsruhe, Germany) for immunohistochemistry or 4',6-diamidino-2-phenylindole (DAPI, Life Technologies) for immunofluorescence, respectively. Visualization was performed with the appropriate VECTASTAIN ABC reagents for biotinylated antibodies and either ImmPACT DAB or VIP Peroxidase (HRP) Substrate (all from Vector Laboratories) according to the manufacturer’s protocol. For immunofluorescence Alexa Fluor 488 and 555 conjugated secondary antibodies were used (Life Technologies). Staining for collagen (Picrosirius Red) were assessed under polarized light. Pictures were captured using a DM4000 B microscope with a DFC320 camera for immunohistochemistry and a DFC350FX camera for immunofluorescence and QWin software (all Leica Microsystems).

For this application, we mainly followed an already established protocol ("method 1" in [63 ]). In the case of solid emulsions, a small piece with dimensions 6.5 mm × 4.1 mm × 3.0 mm was cut out and glued into the hole of a copper carrier with Tissue-Tek^® O.C.T.TM (Sakura Finetek Europe B. V., Alphen aan den Rijn, The Netherlands). In the case of liquid emulsions, the emulsion was homogenized by an ultrasonic transducer UP200S (Hielscher Ultrasonics, Teltow, Germany) at an intensity of 85% for two minutes. After homogenization, the liquid was added in a hole of a copper carrier until the formation of a bulge on the surface of the carrier. We used the transfer unit/preparation chamber Emitech K1250X (Quorum Technologies, Laughton, UK) (the sublimation time amounted to 3–4 min at −95 °C to −80 °C) and the SEM Evo LS10 (Carl Zeiss Microscopy GmbH, Jena, Germany).
The resulting cryo-SEM images were analyzed by single measurements of about 20 randomly distributed droplets by using the digital image processing software AxioVision (v. 4.6.3, Carl Zeiss Microscopy GmbH).