Régulation des populations de Nématodes gastro-intestinaux ...

Résultats
for 1h at 37°C with 200 µL of 5% skimmed milk PBS-T (PBS-TSM). Subsequently the PBS-
TSM was discarded and the plates dried, before adding 100 µL of serum diluted in PBS-TSM
or undiluted mucus to each well for 1h at 37°C. After three washes with PBS-T (the third
being of 5 minutes), the plates were subsequently incubated for 1,5h with 100 µL of Donkey
anti-sheep IgG HRP (Sigma, reference A3415) (IgG determination) or for two periods of 1h
with the first (Mouse IgG1 anti-IgA bovine/ovine, Serotec, reference MCA628) and the
second (Goat anti-mouse IgG1 HRP, Serotec, reference STAR81P) conjugates separated by
three washings in PBS-T (IgA determination). Finally, the plates were washed three times
with PBS-T and 100 µL of 2-2’-azino-bis (3-ethylbenylthiazoline-6-sulphonic acid, ABTS) in
100 mM citrate buffer (Sigma, reference 104.4) containing 0.01% H2O2 was added to each
well. The colour was allowed to develop for 1h at 37°C. To stop the reaction, the plates were
placed 15 min at 4°C. The optical density was measured at 405 nm using a Microplate Reader
(System Dias, Dynatech).
2.5. Histology
At necropsy, two tissue samples from duodenum (between 50 and 100 cm from the
pylorus) were taken ; the first one was fixed in a 10% formalin for conventional histology and
immunohistochemistry (IHC), and the second was frozen at –70°C for frozen IHC.
2.5.1. Conventional histology and IHC with paraffin-embedded samples
Duodenal tissue was embedded in paraffin wax and tissue sections of 3 µm were
mounted on glass slides. Eosinophils and globule leukocytes were counted on
haematoxylin/eosin stained slides, whereas mast cells were counted on Giemsa stained slides.
Immunohistochemistry was performed using antibodies already described for cell
phenotyping on paraffin-embedded tissues in sheep (Lacroux et al., 2006). This antibody set
allows specific labelling of macrophages/monocytes (mouse anti-human CD68 antibody,
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