biochemical and haematological profile - Universitatea de Ştiinţe ...

Universitatea de Științe Agricole și Medicină Veterinară Iași
of hatched embryos represented approximately 60% (45 to 75%) of the collected eggs. The
vesiculated eleuthero‐embryos were transferred in 12 wells microplates (5 eleuthero‐embryos
in 2ml medium/well). Negative controls and MCLR media were not changed during the 48
hours experimental procedure. The vesiculated fish larvae being fully autotrophic, no food was
given during the time course of the 48h experimental period. Non‐invasive observations of
eleuthero‐embryos behavior were performed and eleuthero‐embryos were handled in
accordance with European Union regulations concerning the protection of experimental
animals.
Histopathology process
At the end of the immersion experiment, surviving hatched embryos treated or not (control)
with MC‐LR were fixed in buffered formaldehyde solution 10% (v/v) for 24h. Fixed embryos
were then clarified and dehydrated in successive baths of ethanol (70% and 95%) and butanol,
and finally transferred into baths of liquid paraffin at 56°C. They were individually oriented and
embedded into blocks of paraffin wax. Transverse sections (3,5µm thickness) were hydrated in
successive baths of ethanol (100% and 95%) and routinely stained with Hematoxyline‐Eosine‐
Saffron (HES, Sigma‐Aldrich, France). Microphotograph observations of histological transverse
sections were carried out with an Axio‐Imager Z1 Zeiss microscope at various magnifications.
The all embryo being cut, an constant anatomical level was chosen in the pectoral fin region to
perform observations on intestine and liver.
Immunohistochemistry process
Sections of paraffin‐embedded tissues were deparaffined in toluene and hydrated gradually in
ethanol (100% and 95%), washed in distilled water and then in 10mM phosphate buffered
saline (PBS, Sigma‐Aldrich, France). Tissue sections were microwaved in 10mM citrate buffer,
pH 6.0 for 30min (350w microwave oven). Two different primary anti‐MCLR monoclonal
antibodies (Alexis Biochemicals, Switzerland) were tested: AD4G2 which recognizes all
microcystins because of its Adda specification and MC10E7 which recognizes all 4‐Arg
microcystins. The immunoassay was routinely performed with the NexES system (Ventana,
Tucson, USA). As positive control, we choose an adult medaka fish liver, which proceeded from
a gavage experiment with MCLR (Mezhoud and al, 2008), and tissue sections proceeded
without primary antibody were chosen as negative control.
Transmission electron microscopy
Embryos were preserved in 0.5% glutaraldehyde in 2% paraformaldehyde solution for
transmission electron microscopy study during 24 hours and then, washed three times in
Sorensen phosphate buffer (0.1M, pH 7.4) in three successive 10min baths; finally embryos
were dehydrated in ethanol (50°, 70°, 90° and 100°) by three baths at each step of dehydration
and the embedded in a epoxy mixture (Spur). Medium and ultrathin sections were sliced with
diamond knives (Diatome) on a Reichert‐Jung Ultracut microtome. Ultrathin sections on grid
were then observed by transmission electronic microscope (Hitachi H 700S) with a camera
(LCD, Hamamatsu) at various magnifications.
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