[Histonet] Re: Processing whole rat heads

[gayle callis]

You will need to totally fix the heads over a longer period of time.
Perfusion is ideal if you can to that, followed by immersion fixation for a
week.   Otherwise, simply remove the head and immerse into a very large
volume of NBF.    If you only want to see is the skull and brain, you can
disarticulate and remove the lower jaw carefully so as to not damage the
skull.   We removed the skin and ears by stripping the skin from back of
head towards the nose  leaving the flesh around the end of nose(nares)
intact, before immersing in NBF.    The eyes can be left in and are good
landmarks during microscopy.  

 

Immersion fixation should be done for 7 to 10 days, longer being better.  I
suggest changing the NBF after the first or second day, and then again after
several days to replenish the aldehyde component.  You want total fixation
in order to protect the soft tissues and nuclear staining from effects of
acid decalcification.    

 

Decalcification can be done with 15 to 20% formic acid.  If you are not
planning to do any immunohistochemistry or special stains affected by the
acid decalcifying agents, a commercial HCl based decalcification will speed
up decalcification.  Change the decalcifying solution daily, and do
decalcification endpoint testing to prevent over exposure to either formic
and HCl acids i.e. "over decalcification' that damages nuclear staining,
especially H&E.   The main thing is to fix and decalcify the teeth and inner
ear bones completely as these will take longer to decalcify than the thinner
skull bones.    I suggest suspending the heads during fixation and
decalcification so these solutions surround the head completely.  Large
nylon biopsy bags work nicely, can be suspended with strings into solutions.
Although we did not attempt to remove teeth and if you don't need to see
them, this could be done after decalcification  and the water rinse.  Cut
off incisors  with a very sharp cuticle scissors (yes, the ones found in
nail salons and very cheap at Walmart).  Be careful to not disrupt the bone
when doing this.  The molar and other teeth will probably have to remain
since pulling those teeth will disrupt bone/soft tissues.    

 

After decalcification is complete, and if waiting for other heads to finish
decalcifying, rinse the heads in running tap water for 4 hours and them
transfer to either NBF to stop the decalcification.    One can transfer to
70% alcohol which is then the first solvent used in the processing schedule.
Do not go back into NBF after storing in 70% alcohol.        

 

Your processing needs to be extended considerably with the whole rat head.
Automated processing should be done with  vacuum and pressure.  You may end
up having to process for 2 1/2 to 3 hours per station with ambient
temperature (no heat added) and vacuum on, depending on the processor.  I
suggest trial runs with normal rat heads to optimize processing.  This will
require a dedicated schedule in order to have good dehydration, clearing and
paraffin infiltration with a minimum of three paraffin changes, no less.
Harder paraffin is desirable but not necessary i.e. Tissue Prep 2 or an
equivalent,  to support the head during sectioning.   Heat added to
processing only dries out rodent tissues/bones excessively.    We processed
heads in large nylon bags but hold the bags down securely so they don't
float while in the processor chamber.   We used a  curved hemostat to clamp
tops of bags and hold them down inside the metal processor baskets.  There
will be a certain amount of shrinkage of soft brain from bone due to
processing solvents.   

 

We embedded in the largest Peel a Way mold (I have photos of how to do this
if you need them, and will send privately).   This way you avoid those
annoying, unstable,  large mega cassettes the move around in the block
holder, and probably too small for a rat head anyway.   Before embedding,
put labeled cassette backs and largest Peel away molds in embedding center
to be hot during embedding procedure.   Fill hot mold with melted paraffin,
embed head and anchor head on cold plate to prevent floating up/maintain
orientation.   The head orientation is for mid sagittal cuts.  Working
quickly, push the hot, labeled  tissue cassette(with lid removed)  down to
but not quite touching the side of the head.   It takes practice but the hot
cassette can be pushed down evenly.  We use two thumb dressing forceps to do
this, and could level the cassette fairly easily simply by eye.  Just get
down to eye level to see this while the paraffin is hot.   The hot peel away
mold expands outwards without tearing apart when adding the cassette back.
Be gentle!     This embedment results in a block that doesn't extend
excessively from block holder  and avoids unnecessary/unstable mechanical
forces during sectioning.  The regular cassette back (in horizontal
position) fits snugly in a block holder for stable sectioning.  Trim away
excess paraffin from back, sides and ends of cassette to fit in holder
evenly.    If you try to orient the block so it is on vertical axis rather
than horizontal,  your sectioning will be more difficult., fewer sections in
ribbon for flotation.    The top of skull is rounded so that should take
care of trying to embed the skull at an angle, or you can try more of an
angle if it works for you.    The old fashioned way is to lay each ribbon on
a flat black construction paper, cut the section from ribbon, float and pick
up each section so as to NOT lose a serial section.  This way you can have
many ribbons laying out in sequence before floatation.  Tedious but
accurate.    

 

A regular rotary style  paraffin microtome should work without problems.  5
to 7 um should be adequate.   Overly thick sections will curl up, making
them hard to flatten on water bath.  To section, you should mount the block
in holder so the edge of the blade passes through top of the skull first and
those tough, harder teeth last.  Teeth and inner ear bones will be the
hardest thing to section.   I strongly suggest using high profile disposable
blades for stability as these are just as sharp as low profile.   Doing
serial sections will not be a simple task but certainly possible, requires
practice and patience.   Mount sections on plus charge slides, drain water
off well and dry sections FLAT at 37°C to 40°C for several days.  Do NOT dry
in a hot oven.  Bones, teeth and brain should flatten and adhere well
without excessive drying heat.  Make sure the blade is always sharp, changed
frequently, and soaking not excessive.  You may want to soak the block face
with a gauze wet with cold water with icing the wet gauze frequently while
the block mounted remains in the block holder so you can pick up all
sections.  After a each soak, you will have to back up the block a tiny bit
in order to get the next section in the series.    

 

For staining, avoid overly vigorous rinsing as the softer brain can still
dislodge from bone and/or fold over.    

 

Good luck and send photos to histonet when you have results.      

 

Gayle Callis

HTL/HT/MT(ASCP)