Jb 4 plus

After euthanasia, tibiae were explanted and were fixed in 4% paraformaldehyde (PFA). PFA-fixed tibiae with implants were partially decalcified for about 6 h with a rapid decalcifying formic acid/hydrochloric acid mixture (Decalcifying Solution, VWR) or for about 2 weeks with neutral EDTA solution, and embedded in paraffin wax. Sections (5 μm) of the bone–implant interface were stained with hematoxylin and eosin (H&E), Masson’s trichrome, tartrate-resistant acid phosphatase (TRAP), and gram stains. Implants were embedded in glycol methacrylate (JB-4 Plus, Polysciences) following the manufacturer’s protocol and sectioned. Cell lineages were identified with the help of Dr. Spector, Dr. Bronson, and Dr. Padera.

For analysis of the mutant phenotype, embryos were fixed overnight in Bouin's fixative and embedded in JB-4 Plus® resin (Polysciences). 3 μm sections were cut and stained with toluidine blue.
For assessing expression domains of certain transcription factors following in situ hybridization, embryos were embedded in Paraplast® Plus and sectioned at 10 μm. Other embryos were embedded in 2% low melt agarose in 0.1 × MBS and vibratome sectioned at 30 μm.

Samples imaged in whole-mount were dissected using #5 Dumont Tweezers from the individual and intestines were mounted on a slide in Vectashield (Vector Laboratories).
Samples processed for histology by infiltration and embedded in glycol methacrylate (JB4 plus; Polysciences) or dimethysulf-oxide infiltrated paraffin (McCormick Scientific) for 4 hr at 60°C. Sections were cut on a Leica RM2135 microtome.