Hematoxylin and eosin

The digestive system of G. pyloalis fifth instar larvae after being fed on treated leaves from third instar onwards were dissected out under a stereomicroscope (Olympus Japan) in isotonic ringer saline. The dissected digestive system were immediately fixed in Buin’s fluid for 24 h. Then washed first in tap water and then distilled water. They were processed for dehydration in ethanol grades (30, 50, 70, 90% and then absolute), and the paraffin was used for embedding. The sections were cut at 5 μm thickness by a rotary microtome (Model 2030; Leica, Germany). Routine staining by hematoxylin and eosin (Merck) was used. Photos in control vs. treatments were taken whenever necessary under a light microscope (M1000 light microscope; Leica) equipped with an EOS 600D digital camera (Canon, Japan). The ovary of 2 days old adults were dissected in the similar way and processed as described for gut. However, the sections were cut longitudinally.

Five broilers were ethically sacrificed (cervical dislocation technique) from each experimental group on d 14 and d 28. After immediate dissection of the sacrificed broilers, carcass weight, giblet weight, abdominal fat weight, length, and weight of different gut segments were measured. Segments (1–1.5 cm in length) of the small intestine (duodenum, jejunum, and ileum) and large intestine (cecum and colorectum) were collected and flushed with phosphate-buffered saline (PBS) to wash out the digesta. Collection site for each segment: Duodenum–from the midpoint of the ascending loop; Jejunum–from the mid-region between the entrance of the bile duct and Meckel's diverticulum (MD); Ileum–from the midpoint between the MD and ileocecal junction; Cecum–from the blind end of the right cecum; Colorectum–from the midpoint between the ceco-colic junction and rectal opening.
Intestinal segments were fixed in neutral-buffered formalin (10%) for histological study. Tissue samples were then dehydrated using rising grades of ethanol (Merk, Damstadt, Germany), cleared using xylene (Merck, Damstadt, Germany), and paraffin-embedded. Five micrometer thin tissue sections were cut from the paraffin-embedded gut tissues and finally, Hematoxylin and Eosin staining (Merck, Damstadt, Germany) was performed for histomorphometric investigation.

The skin grafts were fixed in 4% paraformaldehyde and embedded in paraffin. The blocks were sliced into 5-µm sections and stained with anti-mouse CD3ε (A0452, Dako, Glostrup, Denmark) or anti-mouse Iba-1 (019-19741, Wako, Osaka, Japan) as the primary antibody, and then with anti-rabbit IgG-horse radish peroxidase (K4003, Dako) as a secondary antibody. Antibody binding was detected with a chromogenic substrate for horseradish peroxidase. The samples were counter stained with hematoxylin and eosin or Kernechtrot (Merck Millipore, Billerica, MA, USA).

For histopathological evaluation by light microscopy, the corneas were fixed in 10 % neutral buffered formalin for at least 24 h and trimmed along the whole diameter (2 stripes of 3–4 mm width). They were histotechnically processed with a standard method for light microscopy and stained with Hematoxylin and Eosin (Merck KGaA, Germany). Histopathological findings were assessed in the epithelium based on the depth of injury by using a standard semi-quantitative grading system (from 1 to 5) that is related to the extent of affected cell layers beginning from the corneal surface (squamous cell layer) down to the basal cell layer. This depth of the injury has been proposed as a predictor of the severity and reversibility of effects [41 –43 (link)] and has been taken up in an OECD guidance document related to the BCOP assay [44 ]. In the stroma, tissue swelling and keratocyte changes were evaluated. These findings were summarized in a so-called Histopathological Score of Irritation (HSI) assigned for each cornea ranging from 0 = no irritation to IV = severe irritation (cf. Kolle et al. [45 (link)] for further details). HSI IV was assessed as ‘severe irritation’; HSI I, II and III were overall assessed as ‘non-severe irritation’; HSI 0 was regarded as no irritation.