You may want to try using Adult Rat Brain Slicer Matrix, available commercially, along with the rat brain atlas.

http://www.zivicinstruments.com/brain-slicer-matrix-rat-adult-1mm-coronal-brain-slices-section-x-acrylic.html

Palkovits M and Brownstein M J (1983) Microdissection of Brain Areas by the Punch Technique, in Brain Microdissection Techniques (Cuello A C, ed), pp 1–36

Laser Microdissection on cryostat slices...

I suggest to call the request to Giuseppe Luppino university of Parma. He is a very good anatomist.

berst

leopoldo

To get enough sample, the most appropriate in your case would be punches from 300 um thick frozen slices (Palkovits technique)- e.g. Kitabgi P Neurosci Lett. 1991 Mar 11;124(1):9-12.

Amygdala is tricky and you may need to punch it out. But isolation of striatum and PFC is very easy indeed. Take the brian out and put it on a buffer soaked filter paper with top of the brain pointing upwards. Then using a a watchmaker forceps and a fine spatula open the brain out it out from the midline so that the cortex peels away - like peeling a banana - from the underlying nuclei. . If done carefully and properly, then you will see on each side of the brain two largish ovoid structures. The rostral largest blob is the striatum and the caudal one is the hypocampus. Then all you need to do, using a fine pair of forceps pinch out the structures. The PFC could be got at after you have taken out the striatum/hypocampus. Good luck.

Free-hand is often a good first approximation. The technique you settle upon depends on your anatomical knowledge, the amount of tissue you need for your procedure and the anatomical specificity and reproducibility you need. For example, even such a small structure as amygdala has subdivisions with clearly differentiated connections, thus probably slightly different chemical content and function... see the work of G.D. Petrovich, L.W.Swanson and others. Larger structures such as striatum and prefrontal cortex may also have undiscovered differences within their anatomically defined three dimensional borders.

The rat brain slicer mentioned above may well afford you consistency needed if you have the option to choose same-sized brains for all experiments. Be aware that cryostat section consistency is dependent on ability to reliably place tissue in the same plane for sectioning, and cryostat sections are usually very thin, usually only including a few cell layers. This could be an advantage or not, depending on volume of tissue needed for your assay. One obvious advantage of using cryostat sections may well be ability to process adjacent sections for immuno- or hybridization- histochemical and cytoarchitectonic analysis.

All of the answers above are suitable for the issue. The think is what instruments do you have in hands to help out? I would say that laser microdissection is the best choice but if you dont have the equipment punch will do the job perfectly.

Good luck with your experiment

If you do the cryostat punches, you can buy a relatively expensive neuropuncher that has to be cleaned between animals/areas or you can get disposable blunt-tipped needles and push the sections out with air from an attached syringe. Starting with a thick section will make getting enough tissue easier and you can punch right on the knife if you keep your puncher frozen in the cryostat chamber (otherwise the sections thaw inside). If you are unsure of the anatomy, though, you can save thinner sections frozen on slides in a series so you can stain one series to find your regions on adjacent slides. You have to be careful to keep the ones to be punched frozen.

Dissections for brain homogenate and cryostat sections are 2 different methods.

For all procedures use a brain atlas for guidance of the brain areas.

For brain dissections you have to be fast to avoid tissue degradation. Immediately after sacrifice (lethal anesthesia and decaptation) you put the whole brain over a cold (using ice under) Petri dish.

For PFC dissection: discharge the olfactory bulb, and cut a coronal slice of approximately 2mm bellow the olfactory bulb.

For striatum dissection: after you have cut the PFC separate the 2 hemispheres (like opening a book) using tweezers. Thus, in the interior part of the cortex (like the 2 open pages of the book) you will find kind of oval structures in each middle-inferior part, those are the striatum in each hemisphere. You can remove it using tweezers.

I have no experience dissecting amygdala, however, for any brain dissection I would recommend previous consultation of a brain atlas (3D are even better).

After dissection you have to immediately freeze the structure in liquid nitrogen or put it in tubes and freeze in dry ice. Storage in -80 freezeer.

For cryostat sections, the better way to preserve the brain is by intra-cardiac perfusion of the rat (there are many different protocols). After dissection of the whole brain, leave it immersed in the fixative overnight. The day after immerse it in 30% sucrose (for cryoprotection of the brain cells). After 2 days at 4 degrees C, you can cryosectioning the brain according to the atlas coordinates for each brain area that you are interested.

Good luck!

I hope it helped!

punch biopsy from a frozen section works great, and so is laser micro-dissection. The latter however requires lots of expensive equipment. When doing punch biopsy take thick slices (100-200um) of the brain containing the region of interest and using an atlas and a stereo microscope find the exact the region.

I have used blunted large gauge needles attached to a syringe with a bit of air in it. The punched region can then be popped into a microfuge tube using the syringe for -80C storage.

Please check an online video available on JOVE website to help you isolate the sepcidied brain areas. JOVE journal of visualised experiments.give the key words

Try this http://www.jove.com/video/1938/the-subventricular-zone-en-face-wholemount-staining-and-ependymal-flow

We have been optimizing the a very similar technique. We want to collect RNA from the supraoptic nucleus of rats. What we have been doing is snap freezing the brain after removing it. Then we section the frozen brain at 300um, then use our MacGyver'd tissue puncher to remove the SON and then eject the punch in Trizol. I just got the qPCR data back and it looks like our technique works. Our lab is not in the best economical status right now so we have tried to optimize this process to be as efficient and cheap as possible. Everyone here has given spot on advise, if you would like a little more detail about our protocol let me know and I can send you some images of our setup. One thing to be aware of if you are trying to isolate nucleic acids from these sections is that it is imperative the brain remains frozen from the second its out of the brain until it hits the Trizol (trizol not RNALater since the later is for fresh tissue). We do this by having a few boxes dry ice on hand and again we MacGyver'd some dry ice stages to keep everything cold. LCM is another option except that as it stands today, it is very costly. Good luck

We have had some success using rapid immunolabeling under high salt conditions ( to preserve the RNA) followed by laser capture microdissection. the approach is neither high through put or guaranteed - but it is very accurate.

I have been doing these kinds of dissection for 30 years now. The answer depends on what brain regions you want to collect, and what kind of analyses you plan to perform. See any of our published articles (e.g., in J. Neurochem. For PFC make a coronal cut right at the rostral boundary of the hypothalamus, and then take which part of the PFC you want. Most of the above answers should be useful to you, but contact me if you need more specific information..

Best is the laser micro-dissection.

If you want good sections...first map the whole area of interest using Stereotaxic technique then use the proper laser dissection and see the slices under microscope...then standardize according to your need...

Please check Kuiqpick

www.neuroindx.com

everything else is not precise (manual) or takes too long (LCM)

I alternate sectioning between 25um and 300um on a Leica 1950 cyrostat. I stain the thin (25um) sections with Cresyl violet and use those to identify ROIs to punch using a 0.5 or 0.75mm punch. I've had great success using this to identify the amygdala in adult and developing rat brain. Just be sure to adjust the roll plate in between the thick and thin sections. Also, sectioning at -11C seems to work the best for adult and P21 tissue, a few degrees warmer for younger tissue (P07 and P14).