Dmp 30

Samples of the jejunum (2 cm proximal to Meckel’s diverticulum) collected from two Ross 308 birds and two LD birds on days 1, 7, 21, and 35, and from the LD group on day 63, were fixed in Karnovsky solution (7.5% glutaraldehyde and 3% paraformaldehyde in phosphate buffered saline), washed in 0,1 M cacodylate buffer (cacodylic acid sodium salt trihydrate, Roth; Karlsruhe, Germany), incubated in 1% osmium tetroxide (Chempur; Karlsruhe, Germany) for 120 min., dehydrated in an ascending series of ethanol and washed in the intermedium propylene oxide (1, 2 Epoxypropan; VWR, Germany). Then the specimens were embedded in a mixture of agar 100 (epoxy resin), DDSA (softener), MNA (hardener) and DMP 30 (catalyst) (all: Agar Scientific; Stansted, GBR). Polymerization was done at 45°C and 55°C, each for 24 hours. Semi- and ultrathin sections were cut on an ultramicrotome Reichert Ultracut S (Leica; Wetzlar, Germany). Semi-thin sections (0.5 μm) were stained according to a modified Richardson protocol [9 ] for 45 seconds on an electric hotplate adjusted to 80°C and checked under a light microscope (Olympus CX 21, Olympus; Stuttgart, Germany) to determine the area of interest. Ultrathin (80 nm) sections were mounted on nickel-grids (Agar Scientific; Stansted, GBR) and examined with a transmission electron microscope (Zeiss EM 900; Oberkochen, Germany) (TEM).

RHS was fixed in Karnovsky solution (7.5% glutaraldehyde and 3% paraformaldehyde in phosphate buffered saline) for 4 h, then washed in 0.1 M cacodylate buffer (cacodylic acid sodium salt trihydrate; Roth, Karlsruhe, Germany) and incubated in 1% osmium tetroxide (Chempur; Karlsruhe, Germany) for 120 min. Dehydration was performed in an ascending series of ethanol. Finally, the models were washed in the intermedium propylene oxide (1,2 Epoxypropan; VWR, Darmstadt, Germany).
Embedding of RHS was performed in a mixture of agar 100 (epoxy resin), DDSA (softener), Epoxy embedding medium (hardener), and DMP 30 (catalyst) (all: Agar Scientific, Stansted, UK), followed by polymerization at 45 °C and 55 °C, each for 24 h. Semi- and ultrathin sections were cut using an ultra-microtome Reichert Ultracut S (Leica, Wetzlar, Germany) and the semi-thin sections (0.5 µm) were stained with modified Richardson solution [39 (link)] for 45 s at 80 °C.
Semi-thin sections were examined under light microscopy Olympus CX21 (Olympus, Stuttgart, Germany). Next, ultrathin (80 nm) sections were mounted on nickel-grids (Agar Scientific). The RHS morphology was assessed with an electron microscope (ZeissEM109, Oberkochen, Germany). Images were taken and processed using Adobe^® Photoshop^® (Adobe^® Systems, San José, CA, USA).

To improve localization of EVs for TEM, the EVs were adsorbed onto an epoxy resin substrate containing colloidal gold particles. The substrate was prepared by adsorbing 15 nm gold particles (Department cell biology, University of Utrecht, the Netherlands) on formvar/carbon coated copper specimen grids and then embedded the gold-coated grids in a thin layer of epoxy resin between two layers of Aclar film and polymerized at 60 °C for 48 hours. The EV suspension was placed on the gold-loaded, epoxy-embedded specimen grids and in 1% glutaraldehyde, postfixated in 1% OsO₄ and stained with 1% aqueous uranyl acetate. The EVs on the epoxy-embedded grids were dehydrated in a graded series of ethanol, infiltrated in an Epon Equivalent (AGAR 100, DDSA, MNA and DMP-30, Agar Scientific, UK) and polymerized at 60 °C for 48 hrs. Ultrathin sections of the embedded EVs were prepared using Ultracut S ultramicrotome (Lieca Microsystems, Vienna, Austria) and a Diatome diamond knife (Diatome, Biel, Switzerland). Images using a JEOL JEM 1010 transmission electron microscope (Tokyo, Japan) were acquired with a Morada camera system (Olympus Soft Imageing System, Münster, Germany).