Dear Jason,

interesting question, not easy to answer in one sentence.

Would be interesting also to learn a little bit more about your exptl.(mouse) concept concerning Fe-loading.

Yes there are a lot of fixation possibilities, especially for the lipids in myelin. It is advisable to think about perfusion fixation of the whole brain. But there are other considerations to be taken into acoount additionally. To be short here, I would like to add here an article by John Kiernan,2007 one certainly could use as a starting point to get some clues on fixation of myelin.

Perhaps I will find other papers in my files which could be of help too, so we'll see the outcome within the next days.... Good luck and best regards, Wolfgang

In addition, I would like to pouint you to an elder posting thread:

https://www.researchgate.net/post/Transmission_Electron_Microscopy_TEM_optimisation_and_myelin_headaches

(2012-2013)

Dear Jason,

we found our standard procedures for diagnostic TEM suitable for mouse brain also. Like Wolfgang Muss I would recommend perfusion of the fresh brain tissue with paraformalehyde as a first measure to stabilize the cells. I could send you our standard operating procedures (although in German) if you like (please provide your institutional emal address). Best, Christian Hagel

As you are new to TEM, I suggest you work with someone experienced in the technology. There are a lot of things to get right, and it is something of an art. First, your fixation methods will differ depending on what you are going to do with the sections. For example, fixation in OsO4 and embedding in non-aqueous resin will preclude immuno-labeling, so be sure your protocol is consistent with your experimental plan. Also, published protocols will not include some important details, such as slicing your tissues with the sharpest razor you can find, and generating slices with very small (

Hi Jason,

I generally use acrolein 3.5% followed by paraformaldehyde 4%, and a post-fixation with osmium 1% just before embedding and processing for EM. This protocol works very well for both the grey and white matter. You can find more information here: http://www.jove.com/video/2021/preparation-of-mouse-brain-tissue-for-immunoelectron-microscopy

I would follow all the steps, except those related to the immunostaining.

Let me know if you have any questions!

Hi Jason,

I would recommend to use the protocol published by Graham Knott in NATURE PROTOCOLS | VOL.4 NO.8 | 2009 | 1153 - this works really well

with best regards

Roger

If you don't really want to use immunolabel or immunoperoxidase later on your brain samples I'd try perfusion fixation with a mixture of 2% paraformaldehyde and 2% glutaraldehyde. Sections should be treated by standard osmium (1%) fixation . If you have no experience with perfusion and/or embedding for TEM, you should talk to a local expert. Most of these substances are extremely harmful, and care should be taken during experiments.

Jason, EM for brain slices can prodice very good data for cellular integrity, this is a very interesting research topic. The brain celss are rapidly exposed to anoxic conditions after death or even after a perfusion with a diluted fixative. One method which in my hand gave good results and I used at the Dept Physiology at UCL London (when I was young) is this: mouse brain was obtained by decapitating the mouse and within seconds the brain was sliced, and the slices were immersed in a fixative (1% glutaraldehyde + 4% formaldehyde in 0.1M cacodylate buffer, a modified Karnovsky’s Fixative ). The sliced were subsequently washed in buffer and after Postfixation

1% osmium tetroxide (which is a good staining method for myel in) embedded in epoxy resin. See the attached page from the journal of physiology.

Good luck!

I didn't have time to read the other responses so this may have already been mentioned. In E.M., the primary fixatives are almost always a combination of formaldehyde and glutaraldehyde. Formaldehyde is a smaller molecule, a mono-aldehyde and it penetrates the tissue more rapidly but fixes more slowly. Glutaraldehyde is larger, a dialdehyde and while cross-linking very rapidly, it is slower to penetrate the tissue, Regardless of the primary fixative used, what achieves the stabilization and staining of myelin (and menbranes in general) is the post-fixation step in osmium tetroxide. You need the help of someone experienced in animal perfusion for ultrastructure and in particular, the safe preparation and handling of osmium; it is extremely reactive and even the vapors can damage mucosal tissues like the nasopharynx and corneas. Don't try this without some experienced assistance.

Perfusion-fix if at all possible; include glutaraldehyde. Acrolein is effective but nasty, we avoid it. It's been said that cacodylate is a better buffer for this than phosphate, but we avoid for toxicity. Some find that including ferricyanide (and possibly sucrose) in the osmium postfix helps.

Coggeshall probably published the largest number of pretty high-mag photos of myelin.

Jason,

Key to good brain morphology is a good perfusion and how you handle the material afterward. We generally use a freshly made paraformaldehyde/glutaraldehyde mixture (typically 2% each, but I have seen other good results with 4%PF and 1% Ga). You may have to try a series. Don't let the total concentration go over 5% for both. We make this fresh in Sorenson's phosphate and add 3 mM MgCl2, and filter. If you do a PBS flush at 37C, don't go over a min, then quickly start the cold (4C) perfusate. You can also pre-inject with heparin to avoid blood clotting. We recommend a slow 1-3 ml/min perfusion rate, 30 min total for an adult mouse. After the perfusion, let the animal sit undisturbed in a cold room for 2-3 hours, then gently remove the brain and place in a large vol. of fixative for a slow overnight rock in the cold room. Do your dissections the next day, small pieces 3mm3. After buffer rinsing (I add 3% sucrose to the rinses following initial fixation), post fix in 0.8% potassium ferrocyanide reduced 1% osmium, buffered for 2-3 hours on ice. This is key to myelin ultrastructure. You can then rinse and en-bloc stain with uranyl acetate (in maleate buffer), then dehydrate and infiltrate with resin as usual. We do a 1:1 propylene oxide:Epon with catalyst, rocking overnight. This is followed by 3 more fresh resin changes, with a vacuum infiltration step (15 psi) in the middle of two slow rocking steps. Cure at 60 C for two days.

sincerely,

Michael Delannoy

Johns Hopkins SOM Microscope Facility

5

Hi Jason,

I also recommend to do a slow cardiac perfusion at this case using gravity or circulator. I usually use the fix solution as equal amount as the animal weight per volume, for example, I use 20ml if the mouse weight is 20 g. 2% paraformaldehyde, 2.5 % glutaraldehyde, 0.1 M cacodulate buffer (pH7.4) for primary fixation (pre fixation) is working well for this purpose. and the cacodylate buffer is working better than PBS or other buffer.

Before starting this fixation, I strongly recommend to inject 0.5~1U/g heparin into mouse heart directly, and then, do a slow cardiac perfusion with Ca++ free ringer, 9.1%NaCl, 0.4% KCl, 0.06M MagCl2, 1.2 % HEPES and 1.8% glucose with 0.04M EGTA (pH7.4) until the liver color change to the white. EGTA is helpful to prevent making a stuck in blood vessel because it is effective to relax the vessel.

After that, I wash the tissues with 10% sucrose in 0.1M cacodylate buffer( pH7.4) and do second fixation (post fixation) using 1% osmium in 0.1M cacodylate buffer for 1~1.5 hours on ice.

It is important that the mouse　and rat physiological osmolarity is slight higher than human. (1% physiological saline concentration is adequate for the rodent but not 0.9% saline)

Good luck!

Hi Jason,

We got really good images from myelin using our standard EM protocol for fixation. It was an intracardiac perfusion with 0.9% saline, followed by 2% paraformaldehyde and 2.5% glutaraldehyde. Heads were removed and postfixed in the same fixative overnight, and then brains were dissected. You can take a look into the supplemental data of this study:

https://www.researchgate.net/publication/255171938_The_Subventricular_Zone_Is_Able_to_Respond_to_a_Demyelinating_Lesion_after_Localized_Radiation?ev=prf_pub

Good luck!!!!

Article The Subventricular Zone Is Able to Respond to a Demyelinatin...

Follow this paper with old Tom Reese's expertise:

Tao-Cheng JH, Gallant PE, Brightman MW, Dosemeci A, Reese TS. 2007. Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain. J Comp Neurol 501(5):731-740

Hi Jason,

I was successful to fix the grey and white matter by 4% paraformaldehyde + 2, 5 % glutaraldehyde. The speed of fixation is desirable should be not so fast and at least 80 ml by mouse. Let me know if you have any questions. Good luck.

Neurons transmit information through their extensions – the axons – and form a complex network of connections, which provides the basis for all information processing in the brain. Analyzing this network under the microscope is one of the biggest challenges facing the neurosciences. Most axons are less than one micrometer thick, some even smaller than 100 nanometers. “The electron microscope is the only microscope with a high enough resolution to enable individual axons lying next to each other to be distinguished from each other,” says Winfried Denk. Despite their minute diameter, axons can become very long and extend from one end of the brain to the other. To obtain an overall picture of a brain, the researchers have to analyze large pieces of tissue.

Dear Jason,

I quickly read the answers of other researcehrs. I agree, that the best method is the intracardial fixation with glutaraldehyde in cacodylate buffer. Before that procedure, perfusion with saline is recommended. After the perfusion an overnigth immersion fixation is also improves the result, and the brain should be removed after proper fixation.

Jason,

I am sure everyone would agree that if you have not fixed the brain tissue well within the first minute or so after opening the chest wall for perfusion, there is no volume of fix, length of perfusion time, or postfixation procedure that can bring back that nice tight ultrastructure of myelin. Being methodic and practiced in your steps from euthanasia to first flow of flush through the heart is best. Similarly, if you use decapitation, rapid removal, slicing, and immersion fixation, the same applies. Myelin is one of the first structures to 'relax' postmortem. Any of the fixative concentrations and combinations, along with buffer solutions, recommended above will give you great ultrastructure of myelin - as long as you get it to the region of interest fast enough before anoxia hits.

This is all nasty dangerous material so be cautious and get help from your Health and Safety Office too.

Dear Jason:

The most fixation methods for usual E.M. observation employed in other tissues than nervous tissues may not work well and you get a lot of artifacts in the ultrastructure. Please try the procedure appears in our pyblication (#1). The main purpose for using EM in this study was to observe the ultrastructure of mitochondria.

For Immunohistochemistry, you may be able to use fixation methods employed for other tissues (#2, 3, 4).

For usual electron microscopy:

# 1. Deficiency of schnurri-2, an MHC enhancer binding protein, induces mild chronic inflammation in the brain and confers molecular, neuronal, and behavioral phenotypes related to schizophrenia.Takao K, Kobayashi K, Usuda N, Miyakawa T, et. al. Neuropsychopharmacology. 2013 Jul;38(8):1409-25.

For immunohistchemistry:

#2. Immunogold staining: Miyanari Y, Usuda N, Shimotohno K, et. al.The lipid droplet is an important organelle forhepatitis C virus production. Nature Cell Biology 9(9), 1089-1097 and cover picture, 2007

#3. Immunoperoxidase staining: Suzuki T, Usuda N, et. al. :Occurence of a transcription factor, cAMP response element-binding protrein (CREB), in the postsynaptic sites of the brain. Mol Brain Res 61(1 & 2) and cover picture, 1998.

#4. Immunoperoxidase staining: Usuda N,et. al.: Differential localization of protein kinase C isozymes in retinal neurons. J Cell Biol 112: 1241-1247 and cover picture, 1991

Nobuteru Usuda

29th January 2014.

Hello Jason,

There is basic method of fixing tissues or cells for Transmission Electron Microscopy After removing brain tissue from mouse, immediately make 2mm pieces & wash with 1X PBS followed by fixing these pieces into 3 % Gluteraldehyde for 30 minutes. Then remove gluteraldehyde & finally fixed these tissues with 2 % Osmium Tetroxide (Take precaution using this chemical. Use in fume hood and put goggle to cover eyes. It is hazardous to eyes). After treatment with Osmium Tetroxide pieces will become black. Embed the same in epoxy resin Araldite, section it on Ultra Microtome, collect the sections on EM coated grids, Dehydrate with series of alcohol grades and stain with Uranyl acetate & Lead citrate to get contrast image. Observe these grids under Electron Microscope at desire magnification to see myelin. filaments. Take photographs. I have done lots of work on studying ELECTRON MICROSCOPY of cancer cells and tumors. I think same method will work for you too.

DR. PRAVIN D. POTDAR

I don't have a protocol, I know this only because I worked beside some very good neuroanatomists a few decades ago. They said the trick with myelin is not so much the fixation (a standard fresh EM paraformaldehye/glutaraldehyde mix) as the dehydration steps, which for optimal myelin preservation should be many and small (5% increases in the organic solvent, the whole process taking days).

Ther best classical method to fix brain tissue for EM study including gray matter, myelin etc. is cardiac perfusion with 2.5% glutaraldehyde (or Karnovsky mixture, i.e. 2.5% glutaraldehyde+4% paraformaldehyde) dissolved in 0.1M phosphate bufer (cacodilate bufer is aslo possible).

Hi Jason

I agree with most answers you got, but would like to add to important experiences. 1) Perfusion fixation should be obligatory

2) You can get a real nice fixation without using glutaraldehyde, just 4% formaldehyde in any phosphate buffer. In my hands that kept myelin very well preserved and off course this fixation is better for a of antibodies. The real impo tant issue is to get the perfusion right.

Cheers

Andreas Brech

Look into Publications of HKP Feirabend (also in Methods). You will find and extended comparison for fixation techniques for brain tissue, with special attention towards myelin fixation.

Had to look references up:

H K P Feirabend, H Choufoer, S Ploeger Preservation and staining of myelinated nerve fibers. Methods: A Companion to methods in Enzymology 15: 123-131 (1998).

see also: Feirabend et al 1994, J Neurosci Methods 55, 137-153.

You will not find a better comparison of fixation techniques for myelin.

Jason,

I am in agreement with Dr. Potdar's earlier response (29th January 2014) . Although my interest has been the neuron, specifically degenerating axon terminals, I have beautiful TEMs of myelin using the technique of which he spoke. The classic protocol citation for this is: Fairen A, Peters A, Saldanha J. A new procedure for examining Golgi impregnated neurons by light and electron microscopy. J Neurocytol 1977; 6: 311–37.

Good luck,

Dr. Tony Carboni

Some more notes.

1) To check whether fixation by perfusion is OK and accomplished touch (time by time) with your finger the neck of animal. You will get surely good fixation just after you feel that animal neck became "as hard as wood". As soon as you feel it you can stop perfusion.

2) Postfixation with osmium tetroxide of tissue pieces after removal of corresponding brain zones is absolutely necessary.

3) I think GA fixation is crucial in order to avoid some fixation/postfixation artifacts that could be appeared afte rfixation only with PAF. In this respect classical Karnovsky mixture (see my first comments) is the best.

Good luck!

Either 2.5 - 3.5 % glutaradehyde in cacodylate buffer / phosphate buffer is not so good, or 5% PFA in PBS + 0.5 glutar in PBS. Perfusion is by far, much better than immersion. Phosphate buffer diminishes the quality of myelin lamellae, cacodylate is much better.

Hello Jason,

there are really well-established protocols for (myelin) fixation for EM, which were established in the 60ties by e.g. PK Thomas, A. Peters, S. Palay. As mentioned in earlier replies it is absolutely mandatory

– to fix by cardiac perfusion,

– to include (freshly prepared, EM grade) glutaraldehyde acc. to Karnovski (e.g. 2% GA + 2% PFA) in 0.1M phosphate or cacodylate buffer,

- NOT to overfix (frequent mistake), i.e. perfuse for 15-20min and prepare the tissue required, immerse it in same fixative to complete a total of 2hrs fixation time incl perfusion), carefully rinse several time in PB.

- osmicate in 0,5% - 4% OsO4 (in ddw), after carefully washing out the PB, concentration is less important than time: do not exceed 15 – 30min.

This will yield nicely preserved myelin.

Jason,

Just trying to add information at least for the / a perfusion fixation: cf.

www.ncbi.nlm.nih.gov/pubmed/2391564

J Electron Microsc Tech. 1990 Aug;15(4):383-96.

Preparation of fetal rat brains for light and electron microscopy.

Aström KE, Webster HD

PS: If you have no access to the article, just write back to me....(pdf,2.91MB)

Good luck.

Embedding material and method are also very important. Freeze substitution gets improved results. LRGold is less useful than other resins for fine details, but is good for immuno

Please download my papers on mouse cerebellum from pubmed home.Castejon OJ. If you need further information please advice. Yours. Orlando Castejon.

Adding a tip to Derouiche´s answer, it is very important to delimitate the brain area you need, and post-fixed only the small part containing the area that will be investigated. In this way, you will assure a good penetration of fixative solution in your target during the 2 h of post-fix, at 4o. C. Best of luck in this hard methodology. Even hard, the results will be the gold-method for your investigation!

Jason,

I would perfuse with a PF Ga combo(ie. 3% ,2%) at a slow rate for 30 min (1-3 ml/min) and let the animal sit in the cold room for a couple of hours. Then I would carefully take out the brain and place it in a good volume of fixative overnight in the cold room slow rocking. I would then dissect out small ROI's (1-3 mm3) and after cold buffer rinse (with 3% sucrose) go to 0.8% KFECN6 (potassium ferro cyanide) reduced 1% osmium, for two hours. The reduced osmium is imperative to seeing good myelin sheaths. Remember osmium does not penetrate well (about 0.5 mm), so make your pieces small.

Then follow a standard dehydration embedding protocol (after en-bloc staining if you wish)

good luck,

Michael Delannoy

Fixation by perfusion with 4% PFA + 2- 2.5% GA ( EM grade) in O.1 M phosphate buffer. Brain tissue postfix in the same fixative at 4% for ON. Crucial tips - thoroughly wash out with O.1 M phosphate buffer 3x20 min.

Osmicate in 2%OsO4 in 0.1M PB for hour

The method works. Good luck.

Hi Jason

The answers are all considerable wisdom, I just like to add that apart from fixing as advise colleagues (perfusion fixation with aldehyde and osmium) you have in mind the ability to process fragments of very thin tissue and delicacy of being able to include in a excelnte resin ...

if you follow the recommendations will get excellent results ...

Regards!

There have established protocol

For immuno histochemistry you may use 4 percent para formaldehyde for perfussion and after that you must put the brain into pfa.

For electron microscopy you must be put it into glutaral dehyde.