Apologize for lengthiness, but the matter is quite complex (or - at least should be considered as being a complex one).

Dear Sharif:

it will depend on which size your brain is  (:-))

(o.k., I know THIS sentence makes a bit  to laugh at or to be a 'joke' and therefore again)

Dear Sharif:  it will depend on the species' brain size you have to prepare for your study (unfortunately that isn't mentioned:  neither in the original question nor in your additional question).

"The commonest primary fixative is 4% formaldehyde solution which will penetrate just 2-4 mm in 24 hours [@20°C].  Small specimens measuring less than 10 mm in diameter will fix by simple diffusion. Larger specimens should be thinly sliced, partly dissected or perfused to facilitate rapid and even fixation. "[cf. e.g.: R D Start, C M Layton, S S Cross, J H F Smith: Reassessment of the rate of fixative diffusion. JClin Pathol 1992;45:1120-1121 as only one possible reference out of many more]

So - roughly spoken - it can be assumed that human (whole) brain after state-of-the-art perfusion fixation  (only with  and without dissection into slices after the perfusion) could be immersed into 4%buffered formaldehyde for optimal postfixation for one to three additional week(s), sliced to - say 2 cm thick  slabs - it will take about 72 hrs and more.

But, or ADDITIONALLY, diffusion rate and penetration rate/ time of the primary fixative is NOT proportional with increasing size (see also article pdfs mentioned below), i. e. areas of "good fixation" in a(n ideally) round(ish) sample - 1 cm in width/diameter - perhaps will be 1 or 1.5mm (approx. 0.6 to max 1mm/hr) only, the following 3 mm tissue depth can achieve a 'fixation' within 24 hrs, the inner core of 2mm in diameter then needs at least (or more than) 48 hours.

For Comparison:

In a round(ish) specimen of diameter 5 cm / 50 mm within the first hour approx. 1-1.5 mm "good fixation properties& good preservation of morphological details", Within the next 23 hrs you'll get fixed additional 2-3mm, the following 24 hrs (= then total fix time= 48 hrs) you'll have fixed additional only further 1.5 to 2mm of tissue, the tissue core left - which at that time (= 48 hrs after start of initial fixation !) has a width of approx. 42 (!) mm diameter can be defined as "nearly unfixed to unfixed) and needs essentially more than additional 2-3 days to become fixed by diffusion / unforced penetration.

From these data you might conclude your post fixing for smaller whole brain sizes (rabbit, cat, rat, mouse, etc.),

BUT: successful penetration depth does not mean "full=optimal fixation" over that tissue depth.

Assuming you meant "ideal time period for postfixation" AFTER a successful perfusion: Due to hopefully successfully fixed vessels and cerebral microvasculature emptied completely from RBC's, white & other blood cells, no thrombi - for human whole brain additional at least 2-3 days ( provided no part of the brain [periphery open to air, not under fixative] dried out anyhow...

There are available several textual sources on the subject fixation-penetration-fixation....you also can read: http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/#c22458,

but there might be a more suitable article (unfortunately no author, no year of pblication not citeable therefore) to be found

@ http://pub.extranet.fsu.edu/sites/safety/safetywiki/Wiki%20Documents/Formalin%20Penetration%20Rate.pdf, the latter citing also: 

http://www.abrn.net/pdf/Formalin%20fixation%20protocol%20August04.pdf : 

1 mm penetration of formalin per hour for first hour, then 1mm penetration per 3 hours for subsequent thickness at room temperature.

Good luck and best wishes.

Hi Srinivasa,

In my opinion it would not be the same. We usually transcardially perfuse a mouse or rat in order to get fixative to the cells as quick as possible before the cells start to breakdown after death. After flushing out the blood with PBS, the fixative is injected and it instantly kills and fixes the cells at the same time. This prevents degradation of the cells which is really important if you plan to use that brain for histology.

In my experience, it takes too long to get the brain out and into fixative, and this delay is going to degrade your cells. Also perfusion with fixative firms the brain tissue, which makes it much easier to remove from the braincase without damage.

If you plan to fix the tissue, I would recommend transcardial perfusion first, and then a post fix.

Good luck!

No, they are not the same; any handbook on fixation from the seventies and eighties will show its difference. Be aware that if you want to do immunocytochemistry the fixative used is related to how the antibody is made.

Thank you for the inputs, I will perfuse and then post-fix the tissue.

I agree they are not the same.

 TC perfusion is vastly superior with respect to penetration of the fixative, and ultimately the fixation efficacy. Also, it effectively removes all blood cells which can give serious auto fluorescence, which is important when using fluorescence labelling. As said by Leslie, it also firms the brain makling it easier to remove.

Definitely both are not the same. TC has more advantages like other people felt, I just want to add that in TC method since blood will be totally removed with cold PBS wash before fixing, in IHC we likely to get very less background. In case, we are suppose to use half of brain for different purposes like Golgi cox / RNA/protein we should not perfuse it then we simply post fix it to be more economic and it works too.

As said before, it's not the same. If you plan to use IHC, you have to perfuse and post-fix (but up to 48h to avoid over fixation). In some conditions, if you use for example human tissue, you have to fix your samples by immersion in fixative solution (as far as you have a little sample your fixation will be better). Is some cases, you have to postfix your sections if you use In situ hybridization (with radioactive probes).  .

You might be able to get away with immersion fixation alone if you're only fixing a small block of tissue, but for whole brain, you really need to do TC perfusion, followed by post-fix. Depending on how you're going to process the tissue after (e.g., cryosectioning for IHC), you may also want to soak in 20-30% sucrose after the post-fix.

Whenever possible, I will always choose transcardiac perfusion instead of postfixation procedures. Perfused tissues always afford a better histological quality and more reproducible results.

Since in former times I have done a lot of perfusion fixations on rat and other small animals I would like not only to add that all of my foreposters are right in that vs. (often we also used retrograde perfusion technique) naturally IS NOT the same and will not exert similar or same results with regard to preservation of structure(s) as well as maintaining other parameters one perhaps would like to study after processing (e.g. only classical histology using paraffin sections, cryo-sectioning of prefixed material, application of classical enzyme- or (even sophisticated) immunohistochemistry, molecular biological methods, etc.). Size of organ samples matters, for sure. Interesting for me is that "fixative instantly  and fixes the cells at the same time" has been used in the reply by Leslie instead of saying perhaps  biological tissue in a condition near to real time properties ("morphological equivalent") compared to the physiological conditions of the organ.

The main purpose of applying "perfusion fixation" IMHO is not only to get rid of blood components (cells and especially RBC'S) out of the vasculature system without disturbing the lively and  vivid organ (which is for sure primarily necessary for studies on the vasculature system), but primarily to get the fixative as rapid as possible to its target:  cells and (in case of brain) matrix / grey&white matter so as to prevent ischaemic and physiological as well as certainly structural alterations of the tissue.

Unfortunately you have neither mentioned the species you will work on, guessing it will be either mouse or rat, nor you said anything about WHAT you are going to examine/evaluate after the fixation of your brain-material. For most of the species used in animal experimental designs there exists a lot of specific literature regarding perfusion fixation (either whole body or single organ systems like brain, heart, kidney, lung, etc. etc). Every technique has its own physical parameters (“flushing solutions and procedure, cannulation”, use of Heparin: yes or no, start and minimal duration of the perfusion fixation proper, pressure, temperature, isotonicity of fixative, using an “expander” like PVP etc., and wash buffer solutions, etc.) one should care about.

Basically: After in situ, followed by excision of tissue components to be examined ALWAYS (not only thinking of subsequent processing for ultrastructural evaluation) a conventional “immersion-fixation” in toto or for smaller slices of the organ system should be applied for several time (either at RT for some hour or over night at 4°C) before processing the tissue samples further. In my Literature collection on I have about 50 articles, which certainly reflect a synopsis over some 30 years of procedures used and if you are going to tell us a little bit more about your task and what you are going to do with your tissue after fixation and processing perhaps I could help with more specific literature.

Just only 3 hints (out of many) for references:

(Brain rat & rhesus monkey) by D L Rosene and M M Mesulam, J Histochem Cytochem 1978 26: 28, DOI: 10.1177/26.1.413864  Online-Version: http://jhc.sagepub.com/content/26/1/28

  (kidney,rat); (cf: http://www.ncbi.nlm.nih.gov/pubmed/779407)  and

  (heart, piglets) by Richard N. Gates, Hillel Laks, Davis C. Drinkwater, Jr, Abbas Ardehali, Alon S. Aharon, Ana Maria Zaragoza, and Paul A. Chang (Ann Thorac Surg 1995;60:1308-11) Online cf: http://www.annalsthoracicsurgery.org/article/0003-4975(95)00645-2/pdf

Regards and good luck.

NB (edited 2016-10-21,02:33:  I do not understand the downvoters opinion....but it might be better NOT to know his name....it could be that he/she knows nothing about and only is huffy because of the lengthiness....so I invite him/her also to downvote my most recently posted reply  #018....regards and compliments, WM

@ Wolfgang H Muss - I am planning to use Rat brain for Ex vivo MEMRI

OK! In this case use transcardially perfusion of rat.  Optionally,you can perfuse 100 ml of PBS before perfusing PFA.  To ensure that your fixation is well done, clamp hepatic vein to optimize more flow of fixative solution in the brain. You could verify the state of the body of the rat after perfusion, it should be rigid.  

Good luck

under my criteria, absolutly no. Is not the same or equivalent

Transcardiac perfusion carries fixative solution to the capilaries and it spreads from them, tissue denatures from inside. In oposite by inmersion the brain gets fixing from outside, difusion speed of FA is not very high and in addition the surface of contact is quite smaller than when you use the capillary network

Hi all, 

Can anyone please recommend optimum perfusion conditions for mouse brain and spinal cord collection. i.e perfusion time, pressure and any other factors that may affect the tissue ultrastructure. A major motivation for using the transcardial perfusion is to enable the removal of the spinal cord. Following this, I plan to immunohistochemically/florescent and histochemically examine the tissue. In the past I have post fixed, transferred to sucrose then frozen at -80 would others have different suggestions?

Regarding cryoprotection, I would strongly suggest to use a buffered solution of 20% glycerin and 2% dimethylsulfoxide (DMSO) instead of 30% sucrose, the latter often resulting in strong shrinkage due to hiperosmolarity. Once perfusion was completed, just simply store the brain and the spinal cord for 48 h in the glycerin/DMSO solution before start cutting the brain. Obtained sections can be stored at -80 ºC for years before use.

Dear Deidre, please look in: Perfusion techniques in biochemistry by BD Ross. Clarendon Press Oxford, 1972. For you very old, but according to me the best for organ perfusion. The first chapter gives you the principles of organ profusion, second is on measuring perfusion.

Can anyone please suggest the ideal time period for post fixing the whole brain in 4% formaldehyde?

Apologize for lengthiness, but the matter is quite complex (or - at least should be considered as being a complex one).

Dear Sharif:

it will depend on which size your brain is  (:-))

(o.k., I know THIS sentence makes a bit  to laugh at or to be a 'joke' and therefore again)

Dear Sharif:  it will depend on the species' brain size you have to prepare for your study (unfortunately that isn't mentioned:  neither in the original question nor in your additional question).

"The commonest primary fixative is 4% formaldehyde solution which will penetrate just 2-4 mm in 24 hours [@20°C].  Small specimens measuring less than 10 mm in diameter will fix by simple diffusion. Larger specimens should be thinly sliced, partly dissected or perfused to facilitate rapid and even fixation. "[cf. e.g.: R D Start, C M Layton, S S Cross, J H F Smith: Reassessment of the rate of fixative diffusion. JClin Pathol 1992;45:1120-1121 as only one possible reference out of many more]

So - roughly spoken - it can be assumed that human (whole) brain after state-of-the-art perfusion fixation  (only with  and without dissection into slices after the perfusion) could be immersed into 4%buffered formaldehyde for optimal postfixation for one to three additional week(s), sliced to - say 2 cm thick  slabs - it will take about 72 hrs and more.

But, or ADDITIONALLY, diffusion rate and penetration rate/ time of the primary fixative is NOT proportional with increasing size (see also article pdfs mentioned below), i. e. areas of "good fixation" in a(n ideally) round(ish) sample - 1 cm in width/diameter - perhaps will be 1 or 1.5mm (approx. 0.6 to max 1mm/hr) only, the following 3 mm tissue depth can achieve a 'fixation' within 24 hrs, the inner core of 2mm in diameter then needs at least (or more than) 48 hours.

For Comparison:

In a round(ish) specimen of diameter 5 cm / 50 mm within the first hour approx. 1-1.5 mm "good fixation properties& good preservation of morphological details", Within the next 23 hrs you'll get fixed additional 2-3mm, the following 24 hrs (= then total fix time= 48 hrs) you'll have fixed additional only further 1.5 to 2mm of tissue, the tissue core left - which at that time (= 48 hrs after start of initial fixation !) has a width of approx. 42 (!) mm diameter can be defined as "nearly unfixed to unfixed) and needs essentially more than additional 2-3 days to become fixed by diffusion / unforced penetration.

From these data you might conclude your post fixing for smaller whole brain sizes (rabbit, cat, rat, mouse, etc.),

BUT: successful penetration depth does not mean "full=optimal fixation" over that tissue depth.

Assuming you meant "ideal time period for postfixation" AFTER a successful perfusion: Due to hopefully successfully fixed vessels and cerebral microvasculature emptied completely from RBC's, white & other blood cells, no thrombi - for human whole brain additional at least 2-3 days ( provided no part of the brain [periphery open to air, not under fixative] dried out anyhow...

There are available several textual sources on the subject fixation-penetration-fixation....you also can read: http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/#c22458,

but there might be a more suitable article (unfortunately no author, no year of pblication not citeable therefore) to be found

@ http://pub.extranet.fsu.edu/sites/safety/safetywiki/Wiki%20Documents/Formalin%20Penetration%20Rate.pdf, the latter citing also: 

http://www.abrn.net/pdf/Formalin%20fixation%20protocol%20August04.pdf : 

1 mm penetration of formalin per hour for first hour, then 1mm penetration per 3 hours for subsequent thickness at room temperature.

Good luck and best wishes.

@Srinivasa Prasad Kommajosyula:  excuse that I did not reply yet to your initial question:  to be very short:

for [whole] brain - depending also on the further experimental proocedere (IHC, morphology, localization of...., etc., etc.) always transcardial perfusion fixation

(with appropriate composition of

i) correct - isotonic - flushing solution [e.g. containing PVP and or heparin to get rid of blood/WC,RBC's, etc],

ii) buffered [primary] fixative, iii) appropriate / right perfusion pressure and

iii) right duration of primary fixation [e.g.~5--10 mins, usually seen macroscopically by color change of skin and "sensing/feeling" a certain stiffness of the skin area, sometimes people say also formation of small droplets of fixative in the nose area]

is best / optimal.... After excision of [whole] brain, usually "immersion" postfixation in fresh buffered fixative over night at 4°C is done. 

Since 4% (buffered FA) penetrates approx. only 1 mm/ hr at RT you won't fix optimally a whole brain only by immersion fixation....(conf. Handbooks of Histology, Animal tissue processing, as well as ancillary/old original literature on fixation....

Thanks a lot Wolfgang,,,, I know I m super late to notice your post,,,,this is really inf and helpful.

thanks!!!

You're welcome...."it is never too late" (or: 'Better late than never')....

Best wishes and best of luck, W.M.