Leica asp300

The method described by Daneshmand et al.^{59 (link)} was used to prepare samples for morphometry analysis. In summary, jejunal and ileal samples were stored in a 10% formaldehyde phosphate buffer for 48 h. Then, the samples were trimmed and processed on a tissue processor (Excelsior™ AS, Thermo Fisher Scientific, Loughborough, UK), fixed in paraffin using an embedder (Thermo Fisher Histo Star Embedder, Loughborough, UK) and cut with a microtome (Leica HI1210, Leica Microsystems Ltd., Wetzlar, Germany) to a slice of 3 μm, placed on a slide and dehydrated on a hotplate (Leica ASP300S, Leica Microsystems Ltd., Wetzlar, Germany). Then, the prepared samples were dyed with hematoxylin and eosin and examined under a microscope (Olympus BX41, Olympus Corporation, Tokyo, Japan). A total of 8 slides were prepared from the jejunal segment per bird, and 10 individual well-oriented villi were measured per prepared slide (80 villi/bird). The average of slide measurements per sample was stated as a mean for each bird. Villus width (VW) was measured at the base of each villus; villus height (VH) from the top of the villus to the villus-crypt junction, crypt depth (CD) from the base of the adjacent villus to the sub-mucosa, the ratio of VH to CD and villus surface area were calculated.

Dissociated cells were casted on a glass slide at a spinning rate of 1000 rpm for 3 min. For cultured cells, they were seeded on 8 well-chamber slide. Cells were fixed with 4% paraformaldehyde (PFA) diluted with PBS for 15 min. For porcine pancreas, tissue specimen was fixed in formalin overnight and then dehydrated and paraffin-embedded in a tissue processor (Leica ASP300S, Leica Microsystems, Glattbrugg, Switzerland); 6 μm sections were cut on a microtome (Leica RM2255). Sections were dewaxed in xylol and rehydrated in a descending series of ethanol solution (99%, 95%, 90%, 80%, 70%, and water) prior to the performance of antigen retrieval in citrate buffer 0.01 M pH 6.0. For immunostaining, cell slides/sections were permeabilized in 2.0% Triton X100 for 3 min and blocked for 15 min at room temperature with protein block solution (X0909, Dako). The cell slides/sections were incubated with the first antibody for one hour and with the second antibody for 45 min at room temperature. The nuclei of cells were stained with DAPI mounting solution (H-1200, Vector Labs.). The primary antibodies used were monoclonal anti-insulin (I2018, Sigma) and the secondary antibodies was horse anti-mouse IgG Texas Red (Vector Labs.). The cell slides/sections were examined using Leica fluorescent microscope, model DMI54000B with a QImaging Retiga-2000RV camera.