Then take out the tissues from the fixed liquid and conduct gradient alcohol dehydration in accordance with the following sequences: 70% alcohol (1h), 80% (1 h), 95% alcohol (twice, 1 h/time), and 100% alcohol (three times, 0.5 h/time, 0.5 h/time, and 1 h/time) (dimethylbenzene
infusion (twice, 1 h/time); 65[degrees]C paraffin infusion for 3-4 h).
(DMB) was supplied by Chuandong Chemical Reagent Factory, China.
The slide was deparaffinized in dimethylbenzene
followed by rehydration in 80% ethanol.
They were then dehydrated in ethanol and soaked in dimethylbenzene
and then embedded in paraffin.
The samples containing the intervertebral discs and adjacent vertebral endplates from L4L5 were fixed (4% paraformaldehyde; 24 hours), decalcified (20% ethylenediaminetetraacetic acid; pH 7.4; 21 days), dehydrated (gradient ethanol), cleared (dimethylbenzene
), and then embedded in olefin.
The stain of the slices was removed by washing with dimethylbenzene
and dewaxing was followed by immersing the slice in different concentrations of ethanol.
The sections were mounted on poly-L-lysinetreated slides and heated at 60[degrees]C for 3 h before being subjected to two 10-min rounds of dewaxing using dimethylbenzene
. Then, the slides were soaked in absolute ethanol, 95% ethanol, and 70% ethanol for 5 min each.
1/4 of proximal gastrocnemius tissue was collected, fixed by formaldehyde, made transparent by dimethylbenzene
, and embedded in paraffin routinely to prepare serial sections of 5 [micro]m.
On completion of the reaction, the crude product was first dissolved in moderate amount of dimethylbenzene
, and then precipitated with acetone.
In brief, 3.0 g of PPO was dissolved in a mixture of 2methoxyethanol (6 mL) and dimethylbenzene
The following chemicals, ATA, MAn, TBAB, dimethylbenzene
, pyridine, potassium hydroxide (KOH), phenolphthalein, toluene, methanol, ethanol, acetone, and n-heptane were purchased from Aldrich and used as received.