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) _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet