Application suite v4

The 2-μm cross_–sections of jejunal and ileal tissues were obtained after staining with hematoxylin-eosin using standard paraffin-embedding procedures. For each section, ten representative intact villi were selected for morphology examination using Leica DMI6000B light microscope equipped with an image-processing software (Leica application suite V4.2). Villus height (VH) was determined from the tip of villus to the junction of villus and crypt, crypt depth was defined as the depth of emboly between adjacent villi, villus height to crypt depth ratio (VCR) was calculated. The mean value of these ten values represents the final value of each sample.

Alcohol-preserved specimens were processed with hexamethyldisilazane and later card-mounted. Images of the holotype and the egg, larva and pupa of the braconid wasp were taken with a Leica DFC 295 camera attached to a Leica S8 APO Stereozoomtrinocular microscope (Leica, Heerburg, Switzerland). Image stacks were combined into a single image and measurements of the holotype were done using Leica Application Suite V4.2. Images were edited using Photoshop CS8 (Version 6.1) (Adobe Inc.).
Morphological terminology employed in the description follows van Achterberg [24 , 25 ] except wing venation nomenclature which follows Quicke [7 ]. Terms for sculpturing follow Eady [26 (link)] and Harris [27 ].

Tissue samples were fixed in GlyoFixx (Thermo Scientific) for 24–72 h, dehydrated, cleared and embedded in paraffin. Three micron heart sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome and 15 400× magnification images of randomly selected fields covering the ventricular and atrial regions were taken on a Leica DM3000 microscope (40× objective) for quantitative histomorphometric analysis. The base of the heart and major vessels were excluded because of high inherent collagen content. The number of images required for accurate quantification representative of the whole section of myocardium was determined by a stability analysis of measurements based on a range of fields from a minimum of 5 to a maxiumum of 20. An index of cellular infiltration was derived by quantifying the number of nuclei in images of H&E stained sections. An increase in the number of nuclei compared with uninfected controls was considered indicative of myocarditis. A fibrosis index was derived by quantifying the area of collagen (blue pixels) in images of trichrome stained sections. An increase in collagen content compared with uninfected controls was considered indicative of cardiac fibrosis. All images were analysed using Leica Application Suite v4.5.0.

Heart samples were fixed in GlyoFixx (Thermo Scientific) for 24–72 hours, then dehydrated, cleared, and embedded in paraffin. Three micron sections were stained with haematoxylin for 8 minutes, followed by picro-sirius red for 1 hour, then dehydrated and mounted with DPX. Ten 400X magnification images of randomly selected fields covering the ventricular and atrial regions were taken on a Leica DM3000 microscope for quantitative histomorphometric analysis. The base of the heart and major vessels were excluded due to high inherent collagen content. A fibrosis index was derived by automated quantification of the proportion of tissue staining positive for collagen (red pixels), using Leica Application Suite v4.5.0. An increase in collagen content compared to uninfected controls was considered indicative of myocardial fibrosis.

Paraffin-embedded, fixed tissue blocks were prepared and 3–5 μm sections were stained with haematoxylin and eosin as described [19 (link),32 (link)]. For tubulin β-3 immunohistochemistry, sections were subjected to heat-induced epitope retrieval by incubation in 10 mM sodium citrate, 0.05% Tween20 for 30 min at 95°C then cooled and rinsed in distilled water. Sections were blocked with 10% sheep serum and 1% BSA in TBS for 30 min then incubated at 4°C overnight with 1 μg ml^-1 rabbit polyclonal anti- tubulin β-3 IgG (Biolegend) and 1% BSA in TBS. Sections were then washed with 0.025% Triton X-100 in TBS and endogenous peroxidase activity was quenched with 3% H₂O₂ for 30 min. Bound primary antibody was labelled with excess volume of HRP polymer anti-rabbit IgG reagent (Vector Labs) with 1% BSA in TBS for 30 min. Slides were then washed as previously and incubated with DAB (Thermo) for 5 min. Sections were counterstained with haematoxylin and mounted with DPX.
Images were acquired using a Leica DFC295 camera attached to a Leica DM3000 microscope. For analysis of inflammation, nuclei were counted automatically using the Leica Application Suite V4.5 software (Leica). DAB intensity was analysed as integrated density in ImageJ.