For example formaldehyde, glutaraldehyde or acrolein? I am particularly interested to understand whether soluble proteins may be largely relocated during chemical fixation.
Dear Wolfgang Muss, appologies for the link but below I am providing you with another link, hoping that it will work this time. Also I am sending to you a list of references including the names of Baker and Helander as well as Medwar who is a pioneer in the subject. Best wishes and regards, Ahmed
Formaldehyde 1cm/hour. Glutaraldehyde 1mm/hour. Maximun size for Glutaraldehyde fixation is 1cm3.
There is a number of factors affecting quality of fixation including pH, temp, osmolarity, fixation time, and sample size respectively.
Those have to be optimised for the given sample.
You asked for soluble prots- studying soluble proteins, strong (2%) glutaraldehyde fixation is needed to reduce protein extraction.
The penetration rates of fixatives are limited but can be improved.
How?-with co-incubation;
for example, 2% glutaraldehyde fixation for 2 h can produce good fixation up to a depth of 1mm whereas this may be increased to as much as 5mm if 2% paraformaldehyde is also included.
This is related to the fact that paraformaldehyde penetrates tissue much more rapidly, increasing permeability of the sample for more effective glutaradehyde penetration. In practice, preparing sample blocks of
There is only negligible liquid flow during fixation. That means there's no movement to relocate any proteins. There's only diffusion. And the cross-linking by fixatives is the only way to stop relocation by diffusion...
As you likely know, your question has more variables than a simple question might indicate. Penetration rates are impacted by tissue or material type. To offer example we can discuss the extremes. A porous tissue will have faster penetration rates than a dense tissue, and total depth of penetration will be better for a more porous tissue than for a dense tissue. Generally, I have been taught to be, and have experience to be leery of immersion penetration expectations of more than 1mm for any fixative, but this is a tissue dependent issue and trial and error is the best indicator for a specific tissue. Often we have combined both perfusion and immersion (somewhat simultaneously) in an effort to fix deep tissues well, which can be very successful, but timing for the immersion component can be critical. As you discuss, protein drift or loss, and lipid drift or loss can be critical issues, and tissue type can have a huge impact. Pig taste buds, for example, are highly subject to lipid loss. So the speed with which one can get the tetroxide or secondary fixative to the tissue matters. No hold times or extended buffer washes following the aldehyde or primary fixation are tolerable for this tissue type.
The next concern is the speed with which the fixative can fix the tissue. Penetration is one thing, getting the tissue fixed can be an entirely separate variable. Part of the impact for this is tissue type, and part has to do with the dilution effect that occurs as the fixative moves through the tissue. With deeper layers the fixative is less concentrated, so deeper layers take longer to fix and there is more opportunity for protein and lipid drift.
The last concern discussed here would be osmolarity of the fixative. If the fixative does not have the correct osmolarity for a specific tissue type, the tissue can be subject to protein or lipid drift. I have also found that neighboring tissues can be very different for their osmolarity requirement.
*Gluteraldehyde comparatively high molecular weight limits its ability to diffuse into thick specimens, such as tissue sections or embryos. This is further exacerbated by the fact that as the tissue is cross-linked by the fix, its ability to penetrate over time diminishes.
*Formaldehyde does not cross-link as effectively as glutaraldehyde, and for this reason is rarely used by-itself for electron microscopy. However, its small molecular weight allows it to penetrate cells and tissues rapidly, making it a choice fixative for thicker samples.
so generally use a mixture of below combination
1) 0.5% gluteraldehyde/4% paraformaldehyde in 0.1M cacodylate buffer
2) 2% glutaraldehyde/2% paraformaldehyde in 0.1M cacodylate buffer (PBS, HEPES, PIPES).
The penetration rate of a fixing agent depends on its diffusion characteristics and varies from agent to agent. As devised by Medawar it can be expressed as d = K√t, where d is the depth of penetration, K is the coefficient of diffusion (specific for each fixative), and t is the time. 1 In practical terms this means that the coefficient of diffusion (K) is the distance in millimeters that the fixative has diffused into the tissue in one hour. For 10% formalin K = 0.78. This means that your formalin fixative should not be expected to penetrate more than say 1 mm in an hour and it will take approximately 25 hours to penetrate to the centre of a 10 mm thick specimen , i.e. 5 mm ( = 5² hours).
More details are available at :
http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-2-factors-influencing-chemical-fixation-formaldehyde-and-glutaraldehyde/
2% Glutaraldehyde will penetrate about 0.7mm in 3hours at room temperature. After 24 hours glutaraldehyde penetrates to a depth of about 1.5mm.
More details are available in book by M A Hayat excerpts of which are given as attachment.
Penetration of fixatives into tissues, particularly from plants is a major problem for TEM studies. There are several factors, already mentioned, controlling fixative transfer into cytoplasm of cells. Turgidity of cells and air in the tissue also inhibits fixative penetration. Obviously, the cells at the periphery of the tissue are better fixed than the interior ones. Therefore, it is always better to use these surface cells to observe under TEM.
There is an excellent paper from 1978 which would help answering your question. The reference is the following: Mersey, B., McCully, M.E.: Monitoring the course of fixation of plant cells. J. Microsc. 114, 49-76 (1978). I would advise you strongly to read it.
As a rule of thumb for glut we (in electron microscopy at least) assume penetration (and fixation) of 0.5-1 mm for the first hour. With increasing thickness speed of penetration will significantly decrease. For overnight fixation in glut of a rat’s brain depths of fixation was 2-3 mm (Paljarvi, L., Garcia, J.H. & Kalimo, H. (1979) Histochemical Journal 11: 267-276).
An interesting and relevant paper by Coetzee & vdMerwe: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.1985.tb02570.x/abstract
Another publication for your consideration is Reassessment of the rate of fixative diffusion by R D Start, C M Layton, S S Cross, J H F Smith in Journal of Clinical Pathology 1992;45:1120-1121. They estimated the penetration rate of formaldehyde to be 2.4mm in 24 hours. They also include some useful early references.
... and the paper's for free at PubMed Central:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC495013/
Dear Christopher, I am sorry to correct your data given above:
"They estimated the penetration rate of formaldehyde to be in 24 hours. must read: 2 4 mm / 24 hrs.
[cf: open access: http://jcp.bmj.com/content/45/12/1120.full.pdf+html )
best wishes and regards,
Wolfgang MUSS, Salzburg, Austria
Dear Wolfgang - the manuscript clearly reads 2.4mm, not 2-4 mm, in exactly the same way as it reads 0.02% chromic acid, not 0-02% chromic acid. I am only reporting what the authors wrote.
Best wishes
Chris
Dear Christopher....(;-((
I apologize honestly for my fault, I've made the shot without magnifying this little dot in between - which usually is expected to at the base line..., maybe it is time to retire or emigrate... You are right, best regards,
Wolfgang
(-LONG-) Dear George Komis, dear Christopher,
‚A fault confessed is half redressed‘ and therefore as repentance I shall point you to the fixative “acrolein” which unfortunately is not used widely in TEM-fixation perhaps due to its hazardous properties. If I remember correctly this small, highly reactive aldehyde (at least in former times, and rarely in our century) – besides the usual fixative mixtures - was used for (classical) enzyme histochemistry-TEM due to its fast action (diffusion) (e.g. BENDYAN M, 'google' search for < Bendayan and Acrolein fixative > & cf. also ). I have seen some EM-micrograph showing real extrusion of material out of a vacuole structure, where the extruded material seemed to “flow” through a “membranous tunnel” rather than displaying a "simple, transported” vesicle.
In my files I have found also an article (TEM-IEM, brain):
Use of Electron Microscopy in the Detection of Adrenergic Receptors,
by Chive AOKI, Sarina RODRIGUES, and Hitoshi KUROSE in: Methods Mol Biol. 2000 ; 126: 535–563
Methods Mol Biol. Author manuscript; available in PMC 2010 June 8. @ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2882091/pdf/nihms-183851.pdf
They state in their article (citing)
>
(reference 24. for Acrolein:
24. King, L. c.; Lechan, RM.; Kugel, G.; Anthony, ELP. Acrolein: a fixative for immunocytochemical localization of peptides in the central nervous system. J. Histochem. Cytochem 1983;31:62–68. [PubMed: 6187805])
Since I do have a lot of pdf’s on the subject “aldehyde fixatives” which might contain hidden information on “penetration rates” it might be possible that I’ll come back here with additional (ancient) information.
Best wishes, all have a nice weekend,
Wolfgang MUSS, Salzburg, Austria
Dear Wolfgang, thanks for reminding about acrolein. Brief web search shows it is still in use. May be I’ll try it in future.
http://caur.uga.edu/lect4.htm
http://ukpmc.ac.uk/articles/PMC2861341//reload=0;jsessionid=gIu178MWqrMaCqkivS6a.6
There are factors that affect loss of soluble proteins other than choice of fixative. Much of soluble protein loss occurs after tissue harvesting and before fixation. So, to avoid loss of soluble proteins, it is imperative that the tissue be fixed immediately. Also, the tissue should be sectioned as thinly as practical (ideally into sections no more than a few mm in thickness) to increase the likelihood of fixative penetration through the full thickness in the shortest time. If the tissue cannot be sectioned, it can be fixed by perfusion. Also, choice of fixative depends on what post-fixation studies are planned. You might get more direected responses if you say more about how you plan to use the fixed tissues.
Dear George, dear Vladimir, and others, I have compiled some Acrolein-containing Fixatives (cf. enclosed pdf) out of Millonig's Laboratory Handbook 1976 (with References). The "recipes" are short and easy to follow.
Best regards and good luck,
Wolfgang
Hi, There is a calculation you can use to determine penetration rates of fixatives:
d = K√t where d is penetration depth, K is thediffusion coefficient of the fixative ( for 10% formaldehyde this is 0.70) & t is time. I do not know the diffusion coefficients for gluteraldehyde or acrolein.
...and unfortunately I was not able to find any hint on K for GA, and Acrolein, respectively...BTW, the diffusion / penetration depth depends also on the density of structures, as well as buffer vehicle (composition of mixture Fix/buffer), and temperature, as I have seen in some articles / references. So it might not be easy to define a "best" or "optimal" fixative...as usual, "it will depend" (see the major posts above...)
The penetration rate of formaldehyde is largely irrelevant to tissue no more
than 1.5 cm thick.
However, it's NOT 2mm per hour and fixation takes much longer than this!
It's variable. d =K x the square root of time.
K = the coefficient of diffusibility.
Baker determined K=3.6, probably correct and a figure most people accept.
Helander's 1994 data suggests it is at least 2.0 and most likely close to
3.6.
Using K=3.6 in the above formula, the first layer of cells is penetrated at
a rate of over 90 mm per hour.
This slows to 3.6 mm at 1 hour.
It then takes 4 hours to double the depth penetrated to 7.2 mm, a rate of
1.8mm per hour.
At 16 hours, the depth is again doubled to 14.4 mm, a rate of 0.9 mm/hr.
i.e. to double the depth of penetration you must allow 4X the time and so
doing halves the rate!
Your 4mm tissue block would be fully penetrated in less than 1 hour (3.6 mm
from each surface)
What the issue really is, relates to the binding time necessary for
formaldehyde.
A 90% threshold binding, i.e. MINIMAL binding time is 24 hours at 22C and 18
hours at 37C.
It doesn't matter if your tissue is only a few cells thick or 4mm thick!
16 hours is not long fixation, 3 weeks may be, and is certainly NOT
overkill.
The weak initial binding of formaldehyde is freely reversed, 50% in about 12
hours, by the lower alcohols on the processor and the tissue becomes fixed
by alcohol.
Any thing less than 8 hours formaldehyde fixation reduces the IHC for ER
demonstrably and HER2 markedly.
http://publish.uwo.ca/~jkiernan/FixAnti1
Dear Ahmed Mohamedani, your post really interesting if not elucidating. Unfortunately it seems that (despite using Mozilla Firefox brwoser) the link you have given is not available any more ( I got the server-Reply (Western Univ Ontario, Canada: . Would it be possible to get a title or at least a scan of the information you wanted to display? Also it would be interesting for me and perhaps for all others to find out what Baker and Helander (as you introduced them in your post above) have told us as a whole, so could you - please- cite also the refererence(s) for those authors?? Thank you very much, best regards,
Wolfgang
Dear Wolfgang Muss, appologies for the link but below I am providing you with another link, hoping that it will work this time. Also I am sending to you a list of references including the names of Baker and Helander as well as Medwar who is a pioneer in the subject. Best wishes and regards, Ahmed
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