Immunofluorescence: Transversal and longitudinal cryosections of PND (n = 3-5 animals per group), previously incubated in Tyrode solution
alone (control) or in methanolic extract for 5 or 60 minutes (n = 3-5 preparation/group) were collected onto subbed glass slides, permeabilized in ethanol and methanol (-20 [degrees]C, 10 min each), and in 0.1% Triton X-100 in phosphate buffered saline-PBS (15 min, room temperature), rinsed with PBS and incubated overnight in a moist chamber at 4[degrees]C with TRITC-conjugated [alpha]-BTX and then mounted in glycerin-jelly.
In open systems using Tyrode solution
based perfusates, LDH levels tended to be lower (Kietzmann et al., 1993; Friebe et al., 2013a,b), presumably due to the lack of LDH containing erythrocytes, and have been reported to indicate adequate tissue viability of an equine distal limb during perfusion if below 10 U/h (Friebe et al., 2013a).
At normoxic conditions, siRNA-Neg (control group) and siRNA-MCU cardiomyocytes were subjected to 3 h of normothermic hypoxia (H) with an ischemic Tyrode solution
(IT) followed by 3 h of reoxygenation (R).
Standard bicarbonate-buffered Tyrode solution
(equilibrated with 5% CO [sub]2 /23 mM HCO [sub]3[sup]− ) was the same as above, except that the sodium chloride concentration was reduced to 117 mM, and 23 mM NaHCO [sub]3 was added instead of the HEPES (pH 7.40 at 37[degrees]C).
The composition of Tyrode solution
(pH 7.4) was (mM): NaCl 136, KC1 5, [MgCl.sub.2] 0.98, [CaCl.sub.2] 2, [NaH.sub.2][PO.sub4] 0.36, [NaHCO.sub.3] 11.9 and glucose 5.5.
A segment of proximal duodenum (2 cm) was immediately extracted, stripped of adhering tissue, cleaned and placed in 15 ml well containing tyrode solution
[composition (mM): NaCl 137; KC1 2.7; [MgCl.sub.2] 0.98; Ca[Cl.sub.2] 2.0; Na[H.sub.2][PO.sub.4] 0.32; NaH[CO.sub.3] 11.9 and glucose 5.5], pH 7.4 maintained at 37[degrees]C and bubbled with air.
One end of the portal vein was firmly fixed to the bottom of the organ bath containing Tyrode solution
(NaCl 130, KC1 4.7, [KH.sub.2][PO.sub.4] 1.18, Mg[SO.sub.4].
There, the biological preparation, laid and pinned on the chamber paraffin bed with its endocardium surface facing upwards, was superfused by Tyrode solution
(34 [+ or -] 0.1 [degrees]C, 15 ml) buffered and oxygenated with carbogen mixture (oxygen 95%, carbon dioxide 5%, error less than 0.2%).
Two centimeter pieces of ileum were mounted in a 10 ml chamber containing Tyrode solution
(136.0 NaCl, 5.0 KCl, 0.98 Mg[Cl.sub.2], 2.0 Ca[Cl.sub.2], 0.36 Na[H.sub.2]P[O.sub.4], 11.9 NaHC[O.sub.3], and 5.5 glucose, in mM), pH 7.4, 37 [degrees]C and bubbled with air.
The tissue was kept in a Tyrode solution
through continuous bubbling from a mixture of 95% [O.sub.2] and 5% C[O.sub.2], at 37[degrees]C.
The tissue obtained was carefully washed with Tyrode solution
; a 2cm section was dissected and placed in an incubation chamber.
A piece of ileum was removed, cleaned of fat and adhering connective tissues such as mesenteries and placed in a Petri dish containing tyrode solution
. A thread was attached to the top to serve as a marker.