[Histonet] undecalcified bone sectioning

[Chris.Goodall <@t> bristol.ac.uk]

 Hi Thelma, 
here in the UK ,I use LR White Resin, this is produced by the
    London Resin company and the supplier here is Agar Scientific.It`s 
an 
    acrylic resin, not as toxic as methyl methacrylate and more 
reliable in 
    my experience.The only limitation being that you cannot use alcohol 
    based stains because it affects the resin, other than that it`s 
easy to 
    use, and I am also cutting rat and mouse tibias and femurs 
routinely.I 
    use hard grade resin and polymerise at 60C, but you can also cold 
cure 
    in the fridge if you add the accelerator which is good for acid and 
    alkaline phosphotase.If you are wanting to use immunohistochemical 
    methods then I would recommend Technovit 9100 NEW, 
    www.ebsciences.com/histology/methacrylate.htm   although I haven`t 
used 
    it myself because it`s very expensive.
    The method I use for rat tibia is as follows:-
    after fixation transfer to:
    70% ethanol   24hrs
    80% ethanol   24hrs
    90% ethanol   24hrs
    100% ethanol  24hrs
    100% ethanol  24hrs
    100% ethanol  24hrs
    chloroform    24hrs
    100% ethanol  24hrs
    LRWhite resin 48hrs
    LRWhite resin 48hrs


    polymerise at 60C 18hrs
    
      I use the normal tissue cassettes to process the bones and use 
glass 
    tubes to polymerise the blocks. I first put about 5ml of resin that 
has 
    been used for the 1st change of processing into each tube and 
    polymerise it, this gives a base to the blocks to enable you to 
clamp 
    the block firmly in the microtome.I then add about 5ml resin , 
    orientate the bone and polymerise it for 18hrs at 60C. If you coat 
the 
    inside of the tubes with a silicone release solution available from 
    Sigma you can sometimes reuse the tubes.
    I then use a mechanical sander (with filtration, and wearing 
googles 
    and face mask) to sand the block face and the sides of the block to 
a 
    neat square or rectangle , leaving the spare resin "stub" at the 
back 
    to clamp into the microtome block holder.Good sanding saves a lot 
of 
    trimming on the microtome.
    
     I use a Leica RM2155 motorised microtome to cut the sections, it`s 
not 
    possible to cut good sections by hand.I use a 16 cm D profile 
tungsten 
    carbide knife, you will need at least 2, the edge needs to be very 
good 
    and even though it`s a very hard metal, it`s brittle and the edge 
is 
    easily damaged.I use an angle set at 10 degrees on the microtome. I 
cut 
    at 5 microns,at a slow speed, constantly lubricating the knife with 
    water, using a small brush until the desired face is reached, then 
I 
    dry the blockface and apply a thin coating of quick drying clear 
nail 
    varnish....this does not affect staining or fluorescence if the 
animal 
    was given fluorochromes.Let the nail varnish dry, wet the knife and 
cut 
    very slowly, guiding the section over the water using a brush or 
fine 
    forceps, lift the section carefully on to a coated slide on to a 
drop 
    of water, then stain as desired by letting the section float on top 
of 
    each stain, then float into a waterbath at about 56-60C briefly to 
    flatten, pick up on the slide and place damp filter paper on top 
and 
    roll with a glass tube until flat. Dry on a hotplate at least 
overnight 
    before coverslipping, weight the coverslips for about 6-12 hrs.
     Hope this helps.
    Chris.