Can you use a fixative that has more water in the solution?

Dear Muhammad,

the EtOH-Acetic acid (3:1) appears also to be used for Arabidopsis seed (cf.: http://seedgenenetwork.net/media/Arabidopsis_Harvest_and_Fixation_for_LCM.pdf) .

The subsequent use of your cryosections will demand perhaps the specific fixative in A.root tissues which will i) preserve the morphology to a certain extent and ii) will not interfere with your most probably intended methodologies...

other recipes (English)you will find at:

https://pflanzenphysiologie.uni-hohenheim.de/uploads/media/fixierung_einbettung_schneiden_von_arabidopsis_en_01.pdf

or: ftp://ftp.arabidopsis.org/Protocols/EMBOmanual/ch9.pdf

find < II Immunostaining for light microscopy> , p. 9-14.

Cerzainly there will be other recipes for fixation too.....it really will depend on the task you want to work on.

best regards and good luck, Wolfgang

Dear Muhammad,

1) The fixative for morphological analysis sould be in Glutaraldehyde (2%) for overnight

2) the dehydration should be in serial delution of ETHANOL (15, 30 and 50% 1h. for each then 70% for overnight, next, 85 and 95% for 1H for each, and the last step shoud be in Ethanol 100% for 3 or 4 times (1h. for each)

3) for polymerization step, I used the TECHNOVIT 7100. it is more easy

If you want to do cryosections on a cryostat, then fix in 4% formaldehyde in buffer (something like PBS is good) for at least 4 hrs, or a mix of GA and FA. You can simply rinse this out and freeze in your OCT before cryo-sectioning. This is good on root tips for immunowork.

The initial exposure to ethanol:acetic acid is what is causing the shrinkage.

You would only use ethanol for the dehydration steps AFTER fixing in buffered FA or GA if you were then embedding your sample in a resin, such as Technovit for light microscopy.

Dear Mohamed Banora, dear Stefano,

dear Muhammad,

I understand (and really know) that "excellent morphology" can be retained via the full fixation until embedding procedures used in SEM or TEM(Scanning, Transmission Electron microscopy). For such there are plenty resources/recipes available via Google search as well as in PubMed.

For this you can find at least a (one) ResearchGAte posting thread at:

https://www.researchgate.net/post/Has_anyone_done_the_TEM_on_Arabidopsis_protoplast too.

Muhammad may have a special task saying he uses cryosections for laser capturing.

In fact, we do not know exactly what he is doing after the fixation [cit.: "Ethanol:Acetic Acid (3:1) and then replace the solution with 30% sucrose solution. After that I embed them in OCT and use for cryosectioning"]. If he will not use EM for his task then one has to consider another way for doing the prep:

JUST fixing in the acid fixative, then replacing (without any other intermediate steps) into 30% sucrose solution, embedding into OCT (see further note on this following) and cryosectiong....expecting will be impossible. If the target specification ONLY is MORPHOLOGY (LM) then one would recommend the following:

First of all, after the acid fixation (if this is necessary for any reason, since acid fixation always will result in bad / poor morphology) there must be either:

i) further fixation step with buffered (para-)Formaldehyde [(P)FA], or a mixture of (P)FA -Glutaraldehyde(GA), or a mixture of Acrolein - (P)FA-GA followed by,

or

ii) washing step(s) in buffer (3-5 times).

iii) cryopreservation: I guess the imbibition of the A. root tissue with sucrose is done for protecting the tissue against freezing artefacts. This normally is essential in HR-[high resolution] examinations (for EM, e.g. plunge freezing, HPF etc.) and to a lesser extent used in LM-LHistol. preparations (due to the fact that cryosectioning is done at rel. low "cold" temperatures (i.e. down to - 40/-60°C).

Muhammad, you did not tell about the method you are freezing your tissue. But saying you "embed" in OCT, one can guess you freeze your tissues by using the freezing application of a cryomicrotome or by dry ice or production of CO2-snow).

If you do so,

iv) I would incubate the root samples (after buffer washing, see ii) into at least 3 solutions (buffer + sucrose, increasing concentrations...say buffer :+10% sucrose, buffer :+20% sucrose and finally, buffer :+30% sucrose (if necessary). then,

v) use OCT-Compound to "enclose" your sample(s) [Note: OCT does not infiltrate your samples like a resin! will only stabilize (and, yes of course, always improve) your samples for cutting] and

vi) freeze as applicable.

If hunting after HR-solutions, the whole preparation concept has to follow rules established in SEM-TEM-techniques.

best regards and good luck,

Wolfgang

Muhammad,

Probably all substances will demage the tissue in some degree, especially if you use ethanol, it will srank, of course, it is an aggressive dehidrating substance. I work with human tissue for laser capture , I do not know Arabidopsis root. The question is only the rate of water the tissue has. If it has a lot of water, it is better to use a gradient. For example, I collect the tissue sample in liquid nitrogene, and then, I put it to OTC, freeze it, and make a cryosection. Then I put the slides to 70% ethanol - 30% water for the first step 5 minutes, and then 100% ethanol 5 minutes, and then dry. These go on room temperature. I do not know if your tissue will like this combination,but if the tissue has a lot of water it works.

So try to do fixation after the sectioning, and if the tissue is still damaged badly, try to use a gradient. If it is still bad, then try a bit less aggressive fixation-dehidration liquid.

Thanks all for nice discussion. I would like to make more clear about my goals from the experiment.

Infact, not morphology but to obtain good quality of RNA is my ultimate goal. But as I want to isolate RNA from certain infected cells, by using laser capture microdissection, I also need some extent of morphology preserved to differentiate the required cells.

In start, I was using the fixation procedure in a series as below,

Ethanol:Acetic acid (3:1) ----- vaccum infiltration ...... 01 hour slow shaking

Sucrose 10% in PBS .... Vaccum infiltartion .... 03 hours slow shaking

Sucrose 15 % in PBS .... Vaccum infiltration ... 03 hours slow shaking

Sucrose 34% in PBS ... Vaccum infiltartion .... over night slow shaking

All the above steps were carried out at 4 degree celcius ....

Through this above procedure, morphology was very nice, but RNA was degraded a lot.

Later on I discovered that by skipping the first two sucrose concentration steps (10% and 15%), and directly incubating tissues in 34% sucrose produce intact RNA, but unfortunately morphology goes very bad to differentiate the cells which are required to me.

Now I am looking for alternative, where I could get a fair morphology of tissue and good quality of RNA...

I need help in this context

Thanks

We always use Diethylpyrocarbonate (DEPC; diethyl bicarbonate) treated water in order to inactivate RNase enzymes in all of our solutions and we did not have any problem with RNA degradation even if our protocolls are few days long.

Dear Andras Szabo, I always use DEPC (0,1%) treated solutions. DEPC inactivates the RNases which are present in the solutions. But the RNases within the tissues are activated in presence of water and possibly they also work at 4 degree celsius, might be slow at this temperature, but they do function.

In majority of litrarture, I found that people used various ethanol concentartion for dehydrating tissues to embed in Paraffin, but to embed in OCT people used increasing concentrations of sucrose not ethanol....could not understand why?

Dear Muhammad,

apologize if my reply is too lengthy, but it may contain information you need to understand (as of your question above). By no means, please, feel disabused.

Yes, alcohols (especially ethanol) are used for dehydration of tissues (after fixation to be embedded into e.g. paraffin; as well as sections – either frozen (if the sections have to be stained) or paraffin embedded sections after deparaffinization during processing for staining and mounting them under coverglass), this is called “ascending alcohol series” Sometimes you have to do a “descending series of alcohols”,(e.g. if you want to “rehydrate” sections).

Sucrose, often also denoted Saccharose (M= 342,30g/mol, 1M= 34,23%, 0.05M =approx.1,7%, 0.06M= approx. 2.0538%, 1g% rises tonicity approx. 27 mosmol, 0.29M= 300 mosmol) normally is used in two fashions:

1) as an additive for fixatives, like glucose (maintaining either “isotonicity” or making the fixative solution a little bit hypertonic). Usually such an addition will be in the range of 1-2% w/v. Slightly hypertonic fix-solutions have been recommended usually for tissue (block)fixation (whereas fixation of cell cultures should be isotonic, eventually with an addition of calcium for retention/immobilisation of lipids), it will also (as reported) prevent or lower cell/tissue swelling contrary to using isotonic fix-solutions.

There have been a lot of variants of admixtures, most of them = + 2% sucrose (cf. evtly. as a very specific protocol:

RC CURRAN AND J GREGORY in J Clin Pathol 1980;33:1047-1057

http://jcp.bmj.com/content/33/11/1047.full.pdf )

2) Sucrose in higher percentages (-/+ 3 steps with increasing concentrations up to 25/30% Sucrose) is included into freeze-(cryo-)fixation protocols as a cryoprotectant (lowers the freezing point)

(cp. eg. http://www.pnas.org/content/83/8/2422.full.pdf

Stabilization of lipid bilayer vesicles by sucrose during freezing, Strauss & Hauser, Proc. Nati. Acad. Sci. USA Vol. 83, pp. 2422-2426, April 1986 Biophysics

or:

paper of 1984 = access only PPV(pay per view) : http://www.springerlink.com/content/u6640267345n2281/

,

C. Cavallotti, M. C. Mione, P. Napoleone, F. Amenta

The Italian Journal of Neurological Sciences, March 1984, Volume 5, Issue 1, p 99

[or (similar/related content, only PPV too)

@ http://link.springer.com/protocol/10.1007/978-1-59745-324-0_10

Preparation of Frozen Sections for Analysis, by Gary L. Bratthauer

Abstract

The analysis of frozen tissue by antibodies can be accomplished by the quick freezing of a small tissue sample in liquid nitrogen. Super-cooled isopentane can also be used to further the preservation process. Freezing preserves the available proteins in a near-native state for their identification by antibodies raised against naturally folded proteins. The tissues are sectioned onto charged glass slides where they can be optimally fixed in weakly or non-denaturing solutions such as acetone or those that are alcohol-based. Following mild pretreatment steps to allow for antibody use with low background, (the endogenous peroxidase enzyme or oxidative compounds quenched in a hydrogen peroxide solution and available charged sites blocked by incubation in a normal serum solution) the sections are ready for antigen detection.

Another, perhaps interesting article would be:

http://ejournal.sinica.edu.tw/bbas/content/1997/1/bot381-03.html

Bot. Bull. Acad. Sin.(1997) 38, 21-28,

Effect of tonicity and additives to the fixative on ultrastructure of mesophyllous cells in Kandelia candel (L.) Druce (Rhizophoraceae), by Yuan-Hsun Hwang and Shuh-Chun Chen

So, increasing sucrose-concentrations for “FREEZING” (whether it is LN2=liquid nitrogen or “only” OCT compound as an aid to freeze at lower temps than – 196°C) act as cryoprotection steps in lowering the freezing point of encased tissue/particle blocks.

Last but not least I would point you to the following ReseachGate-Thread:

https://www.researchgate.net/post/Cryoprotection_with_sucrose_30_after_neutral-buffered_formalin_NBF_fixation

Regards,

Wolfgang

Again:

Most recently (sent to me today) in the daily issue of BioTechniques I found the following:

"This month's question from the Molecular Biology Forums comes from the “RNA Methods” section" ( @ http://www.biotechniques.com/BiotechniquesJournal/2014/January/Laser-Capture-Microdissection/biotechniques-349457.html ) (not knowing whether you can access the page....if not, I can provide the content).

perhaps with valuable information on the matter "Laser Capture Microdissection and preservation of RNA).

good luck,

Wolfgang

Thanks Wolfgang Muss for detailed reply. This link is not opening on my computer. Can you send me this file please? http://www.pnas.org/content/83/8/2422.full.pdf

I use vacuum infiltration of my tissues in sucrose solution. How can I know that the vacuum infiltration timing is enough or vacuum infiltration has worked? My vacuum infiltration system does not have the gauge to measure the vacuum. I normally give vacuum infiltration for 15 minutes, but I dont know if it is strong enough... Thanks

Dear Muhammad,

as of your request of the STRAUSS & HAUSER PNAS-1986-article find -please- the enclosed pdf (Please respect Copyrights.... personal use only etc.etc.)

Your second question unfortunately must be followed by another question: what kind of vacuum infiltration? There are several, -at least two - methodological possibilities one can do that: i) either water-jet-vac.-pump ii) mechanical (e.g. rotatory/propeller) pump). Once you tell this one we can go ahead....

Hopefully you can access the pdf

Best wishes and regards,

WM

Dear Wolfgang Muss,

Thanks for the answer and pdf file.

I am using the mechanical cavuum pump. It sucks out all the air from the container attached to it. I keep my samples in this container, open tubes, on ice.

Regards

Shahzad

Dear Muhammad,

to be short with this: if you use the mechanical vacuum pump, you should be aware of the fact that one has to think over some physical matters here: You should know about the possibility of "sucking away also water vapour from the sucrose solution..if the mechanical pump will reach - say- 10^-2 mbar...if you do pumping too long (and I guess 15 min. will be) it will alter also the properties of the hydrous 30% sucrose solution and hence also freezing characteristics perhaps. I guess also you will not be able to "see" the specimen when evacuating (not using an exsiccator device made of clear polycarbonate?) ...

Regards,

Wolfgang

PS: principally I feel that the initial question "Is there any solution to maintain tissue morphology?" requests knowledge of a most detailed described prep.-protocol.

Thanks Muss, it means, if I am interested to do long vacuum infiltration, I should replace the solution with fresh one after each 15 minutes.... Thanks

Dear Muhammad,

unfortunately in my honest opinion (IMHO) such a procedure will not be adequate and no good idea.

Every time you will change "fresh solution" in the recipient of the combined gas transfer pump (mechanical propeller pump) you

i) will have to ventilate/areate your recipient,

ii) will insert "fresh solution" which has to be evacuated in the same way as some 15 mins before... the same physical principles apply:

if you don't stop evacuation at a certain point (will depend on surrounding temperature too) you can eventually see "boiling" (it is not physically but due to the negative pressure (effect of

>lowering the boiling point< of water by evacuation (water “boils” at room temperature.... hopefully my determination/definition here in terms of physics is correct).

I have seen such a of solution between 1.1 - 2 x 10^1 mbar (580-600 pascal) which might of course be detrimentous to your sample, but at least will be a problem for your mechanical propeller pump.

You did not say whether you can see your specimen during pumping / evacuation or not. If yes, you should have seen this effect of "bumping [] fluid".

A two-stage-rotatory (propeller) pump will reach that pressure [1.1 - 2 x 10^1 mbar (580-600 pascal)] within 2-3 mins (depending on the working temperature of the pump and on tightness of your recipient).

I would suggest to evacuate your samples [with a mechanical say-two-stage propeller pump ("zweistufige Rotationspumpe") ] only to a certain "safe" pressure, i. e. in the range of 2.5/3.0-4 x 10^1 mbar = 660-700pascal (usually obtained within 1 to maximally 1.5 min) to prevent the "mini-explosion" of the fluid, accompanied by the decrease in temperature of the fluid itself.

When you have evacuated to that point, leave the fluid with your sample at that vacuum as long as you want, hoping that such will aid in "infiltration/penetration" of your fixative into the cell.

Best and good luck,

Wolfgang

Dear Muhammad,

Sorry for my late reply. According to the answer of Wolfgang Muss, I think he answer and give you many of information you need.

But from my experience of plant micro technique.

1. I found most cryo or freezing microtome should embed as same stage of solution.

For example, you want to cut with freezing microtome you should freeze the sample from the beginning. Such as in liquid nitrogen.

2. Anytime you want to fix the sample in solution please make sure that the specimens is filltrated. This mean you need vacuum to remove all gas from the specimen and replace them with fixation solution.

3. Any time you change the surrounding solution in the specimens, let take a 'time' and the 'continue' concentration of solution to fill in the cell. The chemical agent need time to penetrate from tissue into the cell. And do not change the concentrate immidiately. Ex. Using alcohol 15%, 30%,50%,70%,95% respectively. Each time you change the solution, let take a time for chemical penetration into cell too. Then you can create you protocol and technique your own.

Best regards and good luck,

Tip

Carnoy's fixative is not suitable for any delicate tissue or organ, usually it is recommended for probes intended for staining of chromatin or chromosomes. Root is built of strongly vacuolated cells that shrink easily at any disturbance of osmotic pressure - try some fixative used for ultrastructure and dehydrate the probes gradually

good luck :-)