Sections were rinsed in dH2O for 3 min, fixed in 10% buffered formalin for 10 min, rinsed in dH2O for 3 min, dehydrated in a series of graded ethanol (70%, 85 %, 100) 1 min each, and air dried.
For histological studies, specimens of M. blainvillei were anaesthetized in ethyl acetate vapor for a few minutes, fixed in Bouin's fluid for several weeks, dehydrated in an ethanol series, methylbenzoate and butanol, and embedded in paraplast (Kendall).
Next, sections were placed in a 0.5% Cresyl violet solution for 5 mins, dehydrated in a series of alcohols, defatted in xylenes and coverslipped.
The samples were washed twice in distilled water, contrasted with Mayer's haematoxylin for 7 minutes, washed in tap water for 15 minutes, dehydrated in a graded series of ethanol, cleared in xylene, and coverslipped with DPX.
Immediately after the PCR, the slides were fixed in 4% paraformaldehyde for 10 min, washed in Tris HCl buffer saline (TBS) for 3 min, rinsed with distilled water for 3 min, dehydrated in a graded series of alcohol and air dried.
The collected osteochondral samples from the intermediate region of the medial femoral condyles were fixed with 10%% neutral formalin for 24 h, decalcified with 5%% nitric acid for 4 days, dehydrated through a series of graded ethanol solutions, cleared in xylene, and embedded in paraffin.
The samples were rinsed with cacodylate buffer, post-fixed by immersion in 2% osmium tetroxide for 2 h, dehydrated in a graded ethanol series (pre-stained for 30 min with 1% aqueous uranylacetate in 70% ethanol step), immersed in propylene oxide and embedded in Agar 100 resin (Epon-equivalent, polymerized at 60°C).
In brief, the optic nerve near the eyeball were cut, fixed in 4% glutaraldehyde for 48 h, dehydrated with a graded series of ethanol (2 h for each grade), transparented in dimethylbenzene and embedded in paraffin.
Inflorescences from 35S NTAP AtCTF7∆B plants and wild type plants were collected and fixed in 4% glutaraldehyde under vacuum for 2 hrs, dehydrated in a graded ethanol series (40%, 60 %, 80 %, 100steps for 1 h each), and cleared in a 2:1 mixture of benzyl benzoate:benzyl alcohol.
