I have been doing perfusions for a little over a year, but sometimes I don't do any for a couple months, so I still feel new at this. If the body becomes stiff, I always accepted this as a good perfusion. However, I have had some brain tissues that looked poorly perfused when I performed IHC although the bodies were somewhat stiff after perfusion and the livers were no longer red. So my question is, if a perfusion is done well, will the tail always move? If I need tissues that are really well perfused, should I discard mice that do not exhibit any tail movement? Thank you!
Your question is intriguing in that you may be correct in suggesting a bad perfusion if the tail does not move due to caudal muscle contractions... however, the question I have is; what is you main tissue of interest? The brain? If so, the movement of the tail may be completely independent of a good BRAIN perfusion, since they are on opposite sides of the body... If you only need the brain perfused and have no use for the rest of the tissue I suggest clamping the inferior Vena Cava prior to perfusion, allowing a better potential for brain perfusion only, where more fluid can be shunted through the upper half of the body. Also, make absolutely sure you get the perfusion line/needle into the left vetricle and just into aorta, and not through the cardiac septum to the pulmonary vein (i know this may sound obvious, but it's a common accidental mistake). Do you pre-perfuse with saline? This is an important step as the formalin can clot blood, preventing a good perfusion to regions (of the brain) if such a clot forms. I use 4C PBS to flush the body until the 'waste' from the left atrium runs clear, then follow through with formalin/PFA. In a rat this is about 100mL PBS/saline, follwoed by 400mL PFA. A mouse would require much much less volume... Discarding mice is a terrible loss for you and your research and should be avoided at all costs, as you understand, I'm sure. So a good perfusion practice is essential. Please feel free to contact me again regarding this, and I would really like to know of any success in the future and if my 'advice' helped at all. Regards/best!. [email protected]
Hi Jamie, I guess you are right about the tail movement in the sense that if it does not move the formaldehyde probably did not reach the extremities sufficiently, and in this case it is true that the brain will not be well perfused. So, if you think perfusion is a must, Rink-Jan suggestions are excellent. Typical volumes in mouse are 20 ml of ice cold PBS and 20 ml of ice cold formaldehyde in PBS. Classically one should use 4% formaldehyde, 10% might be too high a concentration, which will make reaching the extremities of capillaries difficult due to fixation of the earlier tracts that will block the flow. Besides, after perfusion you should leave the brain O/N in 4% formaldehyde at 4 °C, since the crosslinking reaction of the formaldehyde to form the methylene bridge takes longer time than the first reaction with one amino group.
Still, in our group I have abolished perfusion for the mouse brain, it is a small enough tissue to be fixed by a 48 h treatment in 4% formaldehyde at 4 °C, and we easily cut 20 um thick sections at the vibratome after this treatment. This spares a pretty tough treatment to the mouse without affecting our immunoflurescence analyses. For IHC analyses we freeze the brain unfixed on dry ice, embedded or not in TissueTek (different people have different preferences for the cutting that comes afterwards), cut at the cryostat 8-12 um thick sections and stick them to Superfrost slides. You can keep the sections frozen at -80 °C for a long time and you just need to fix them briefly (10 min) in 4% formaldehyde in PBS at rt.
Another point is the preparation of the formaldehyde. If you use commercial 10% formaline solution it should be no problem to dilute it in PBS, just be sure you don-t have in the bottle too much white precipitate, that is the PFA (see below), which means you are losing titer of free formaldehyde (so your solution will not be 10% anymore, but maybe 9, maybe 8... anyway difficult to estimate) and it might also mean that you have formic acid in the solution since O2 from the air triggers the redox reaction in time. But if you prepare the formaldehyde yourself starting from powder paraformaldehyde (PFA), be sure you don't add any NaOH or KOH. Just prewarm the PBS in a microwave or on a hot plate until it boils and then transfer to a cold stirring plate, start the stirring with a magnetic bar and pour in the PFA. It will dissolve almost instantly. Then just cool it down quickly on ice and sterile filter with a 0.22 um filter. Aliquot and freeze at -20 °C.
Good luck!
http://publish.uwo.ca/~jkiernan/formglut.htm
We used this method to harvest our spinal cord lumbar part for examinating the neural tracker that we injected at distal part of sciatic nerve after implantation of nerve conduit. It stained the regenerated cell bodies. It seems that we managed to get a good structure of rat's spinal cord after we perfused with 100ml of cold PBS followed by 100ml of cold 4% PFA. We inserted the the needle at left ventricle and make a nick at right atrium. Then we observed the redness of blood diluted after perfused with PBS. Then followed by 4%PFA. Few marks as an indication of good perfusion that we observed are discoloration of liver, muscle and tail twitching. This method has to be practiced many times as your rats are really precious to be discarded. Just sharing our experience here. We assumed we successfully perfused the spinal cord as the cord was intact during harvestion and managed to view the morphology of the spinal cord clearly. Thanks
Thank you all for your advice.
I always perfuse first with heparinized PBS (30ml), followed by 30ml 10% formalin and I postfix ON at 4C. Yes, the brain is my only tissue of interest. I have not tried clamping the inferior vena cava, but that is a good idea. Do have any advice on what kind of clamp to use? Occasionally, I will accidently poke through the cardiac septum, but when I do this it is very obvious because the lungs inflate. I have had perfusions where I did not poke through the cardiac septum and the tail still did not move. Is it absolutely necessary for the needle to go into the aorta, or is it sufficient to get it into the left ventricle?
Pietro, I think 10% formalin is the same as 4% PFA. PFA dissolves into water at 37% w/v, and this solution is called 100% formalin. Therefore, 10% formalin is equivalent to 3.7% PFA. Despite the fact that I do a postfix, brains that were badly perfused do not work well for IHC. It sounds very difficult to cut 8-12um sections of an unfixed brain. I imagine it would take alot of practice, but you are avoiding any artifacts or antigen masking due to fixation. However, I have trouble cutting even 20um sections of unfixed brain.
Khairunnisa, do you always see twitching of the tail when you see discoloration of the liver? I have seen very good discoloration of the liver without any tail twitching. Any idea what this indicates? Could it be that the PFA is reaching more proximal areas while not reaching more distal areas?
Luckily, I am working with wild-type mice right now, so it is not too bad to discard an animal, although I try to avoid it if I can. In the future I will be working with transgenics, so I need to perfect this technique before then.
Jamie,
Try perfusing longer with ice cold Formalin/PFA. After clearing the blood as you are doing, we perfuse 4% PFA in PBS +5mM EDTA for 10 minutes (100ml). You can tell after you dissect the brain if the perfusion went well based on the stiffness of the tissue and lack of blood on the whole brain especially the meninges.
Good luck!
Jamie is correct that 10% formalin is the same as 4% pfa. I use a haemostat/artery clamp to occlude the IVC, but a piece of string as a ligature can be just as effective. It sounds to me you are doing everything correctly, Jamie. Unfortunately it's never a perfect world and things do occasionally go sour..... Such as a bad TC perfusion! I must confess that I've only every performed this on rats.. The principle is the same but in practice much more difficult in mice..... Good luck!
Jamie,
We observed discoloration only at first attempt. After we did perfusion several times, then we observed both. I'm not sure whether if no tail twitching, it just perfused proximally only, maybe you can try harvest and see what's going on the brain structure. I'm still practising and improving the technique. This question is really helpful for me. Thanks to all.
Good luck to you dear Jamie.
BTW, one can easily get good 5-10micron sections using a cryostat for brain embedded in a freezing/embedding matrix such TissueTek or OCT etc. You just need to play around with the chamber and stage temperatures a bit... and that depends on the cryostat itself.. it will take some trial and error. In my experience for brain, which is quite fatty in essence, a very cold temp on both (around -20C) has worked for me ok. Try cutting adipose in embedding matirx as practice for cutting difficult tissue, and you'll see such techniques are indeed possible. You just need to get some practice to gain an experienced hand. (if I can do it, then so can you!!). Also, using unfixed tissue for IHC can be great due to lack of fixative-induced crosslinking (see Pietro Pilo Boyl's answer above), but bear in mind you will still want to rid the brain/tissue of all blood (by TC perfusion of cold PBS), particularity for fluoresence labelling since red cells auto-fluoresce, and the remaining blood plasma may play havoc on your immunogenicity of your added antibodies. Just food for thought.
Hi Jamie, sorry for the formaline remark, I admit I'm not very familiar with the medical terms... there is always something to learn! So, fine, your procedures seem really ok and I'm totally with Rink-Jan on both his points: experiments in biology will never be always perfect, and we also experienced a 30-50% failure rate in perfusion, meaning that the tail did not flick and brain was not greatly fixed, with consequent problems in vibratome cutting. Secondly, if you are doing cryosections (which is still not very clear to me) you will be able to cut thin sections even of unfixed tissue at -20 °C on stage and chamber... That's the reason you are freezing, to harden the tissue. If your tissue is not hard enough you will have to lower the temperature (obviously there is a limit, too hard will break because it becomes fragile!)... All our students learn it, I'm sure you can do it as well! Yet, once you fix the brain by formaldehyde perfusion and/or postfixation you will need to permeate it with some cryoprotecting medium to freeze it, typically 30% sucrose, don't you? You can freeze directly with dry ice or in isopentane or TissueTek only unfixed brain, as far as I know... This could be your problem, with 30% sucrose the osmolarity would shrivel unfixed or badly fixed tissue, and I can imagine then the IHC will be a mess...
But honestly, sacrificing the animals by cervical dislocation (no CO2, no anesthetics, no perfusion at all), dissecting the brain, briefly washing it in PBS and either freezing it fresh and cutting cryosections, or fixing it for 40 hrs and cutting vibratome sections, we have no appreciable background in IF, unless the antibodies are very poor (or the staining conditions have not been optimized!)... Still if you really want to perfuse with PBS to remove the blood, I don't think this is a problem for the following snap-freezing and cryosectioning.
Good luck!
Hi Jamie,
When you are performing the perfusion, the best indicator that I have found is too look at the state of other organs during the perfusion. When starting, the first organ to start changing color during a proper perfusion will be the liver. It will start to turn from deep red to pale beige within the first 5mL of PBS. The organ that is the best indicator of the state of the brain from my experience is the kidneys. If the kidneys are still deep red after 10mL and 50% has not changed to pale beige by 20mL, that is usually a sure sign that the brain is not being perfused well. The kidney is not full proof, but it is a fairly accurate identifier over 90% of the time.
I hope that this helps!
Hi,
In our lab we frequently do the perfusion, Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. we used a first saline and then low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.
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