4/23/96

Our lab has been asked to help on a possible project studying mammalian brain tissue. The material will be collected in the field( a few thousand miles fom our lab) and storage times could be up to 3-4 weeks, before the material reaches the lab for processing. I have an excellent fixation method for brain tissue, but its fairly involved and the solutions must be made up fresh; not a real possibility for this field work. In the past, I have had great success, using PIPES buffer for the glutaraldehyde fixation, whenever mammalian em material had to be stored for 3-4 days at 4 degrees C. I would make up the fixative bottle of 4% GTA in 0.15M PIPES, pH 7.35 and send it out. They would add their samples and mail(at @4 degrees C) them to me. I've also had suceess using the GTA/PIPES fixation and then transferring samples to a buffer of 0.1M Na Cacodylate with 0.15M Sucrose, pH 7.4. and holding the sample for a week, at 4 degrees C.

Has anyone had experience in keeping a sample for 4 weeks before final processing. Would it be better to store the sample: 1. in PIPES buffer, after GTA/PIPES fixation?

2. 0.1M Na Cacodylate with 0.15M Sucrose, pH 7.4. after GTA/PIPES fixation?
3. leave in GTA/PIPES fixative for the entire time at 4 Degrees C. ( I worry about the staibility of the GTA over such a long time)
4. Some other method, that works with mammalian tissue.

Any suggestions or thoughts on long term fixation/storage would be greatly appreciated.

Louisa Howard
Louisa.Howard@Dartmouth.EDU

I have had no problems with long term (years) storage of retina in cacodylate buffer (no additives) after glutaraldehyde fixation. Long term (weeks) storage in glut/cacodylate is also not a problem - the tissue doesn't harden excessively - though I've never done it with brain. All at 4 deg C of course.

Diana van Driel
dianavd@eye.usyd.edu.au

To expand upon Louisa Howard's question of long term storage -she is asking about storage in cacodylate buffer.

The question has recently been asked here about long term storage in phosphate buffers. We have a variety of fish, rat and cow tissues that were collected (or will be collected) "just in case" EM may be needed. Some of these tissues have been around for at least 10 years and will likely be here another 10 years.

What are the current thoughts on storage for long lengths of time? Would it be better to keep the tissues in fixative as opposed to buffer? Would cacodylate buffer be preferable to phosphate (or is there something else)? Any special techniques for processing old tissues and getting useable results? How long have tissues been kept around and still been successfully worked with?

Rosemary Harris
rosemary@aec.env.gov.ab.ca

I read the current set of postings about "the best fixative for...", and "the effect of long term storage on..."and see that many researchers are using PIPES adjusted to a pH above 7.2 to buffer their fixatives.

This buffer has a pKa of 6.8 so would anyone like to make a comment on the buffering capacity of this buffer above pH 7.0?

If we have to use a "Good" buffer for fixation, we choose HEPES which has a higher pKa. We get exceptional morphology in cryosections even if we only use formaldehyde. We also used it at a conc. of 300mM recently to fix some shark tissue with similar results. We never did get much from using PIPES.

As for tissue storage, there should be no problem in storing tissue for long periods in fixative, if the samples are to be examined only for morphology. The samples will slowly get harder and more brittle the longer they are in gluteraldehyde but if they are embedded in resin it will not be a problem. There might be some extraction of soluble material with time but that will only help with increasing the final contrast of the specimen.

If the samples are only to be fixed in formaldehyde then the fixative should not be removed. I have heard unconfirmed stories of 10 - 20 year old samples, stored in formaldehyde at RT, being taken off the shelf and successfully labeled with antibodies. I guess it all depends on what the antigens were.

Paul Webster
paul.webster@yale.edu

---Paul Webster wrote:
I read the current set of postings about "the best fixative for...", and "the effect of long term storage on..."and see that many researchers are using PIPES adjusted to a pH above 7.2 to buffer their fixatives.

This buffer has a pKa of 6.8 so would anyone like to make a comment on the buffering capacity of this buffer above pH 7.0?
--- end of quoted material ---

I agree that HEPES would be much better to use at pH 7-3-7.4, given its pKa of 7.4. We use it often in SEM preps. The reason we use PIPES for TEM, is that it appears to cause less extraction in mammalian and plant tissues. This is strictly anectdotal on our part, although Coetzee and van Der Merwe, J of Microscopy135(2) 147-158, 1984, showed that extraction in plant tissue was greater using HEPES than using PIPES.

I'm definitely willing to try HEPES buffer on mammalian brain tissue, as other posters have mentioned using HEPES for TEM, with success. Can I use the same molarity as with PIPES? I currently use a buffer concentraion of 0.1M PIPES pH @7.3, with a gluatraldehyde concentration of 3-4%. Also, I agree with other posters about the toxicity of Na Cacodylate, although the HEPES and PIPES buffers, supposedly have carcinogenic problems. If we work in EM, we have to take all neceesary precautions, when working with these compounds. I think the PIPES or HEPES buffers might be a better choice for field work.

Louisa Howard
Louisa.Howard@Dartmouth.EDU

I must admit I use HEPES buffer, pH 7.2 for my fixatives with great luck and therefore have not been tempted to change it but I think a good argument to use PIPES can be made. PIPES has a pKa of 6.8 and HEPES has one of 7.5. Thus, for a target of pH 7.2, PIPES is 0.4 pH units off and HEPES is 0.3 units. A good rule of thumb is to use a buffer whose pKa is within 0.5 units of the desired pH. HEPES may look to be marginally better than PIPES but fixation solutions tend to acidify during the fixation step so by having a buffer whose pKa is on the low side of the starting pH, one would get increased protection at reasonable buffer concentrations (i.e., the pH would have to drop 0.9 units before being > 0.5 units from PIPES pKa but by only 0.3 units before being > 0.5 units from HEPES pKa). I think both are "Good" buffers in more than one sense and both are definitely superior to cacodylate in many ways (e.g., cost, toxicity, environmental). Furthermore, I believe cacodylate has a pKa of around 6.3! My feeling has always been the continued prevalence of cacodylate buffers simply demonstrates that morphologists are some of the most old-fashioned scientists around and least willing to change. That's my two cents worth.

tphillips@biosci.mbp.missouri.edu
Tom Phillips

PIPES is one of a few buffers suitable for work with cytoskeletal components (e.g. microtubules). It is often used to buffer fixativesfor light & electron microscopy to preserve cytoskeletal structure.

Steve Rogers
srogers@delphi.beckman.uiuc.edu

Hi,
I'd like to chime in on this thread. I have been using PIPES as a buffer, but only in fixations for immunolocalizations. I had used it for morphological studies, but following a conversation with MV Parthasarathy (EM Director, Cornell), I switched back to either phosphate or cacodylate. My original interest stems from the following report: Salema R, I Brandao 1973 The use of PIPES buffer in the fixation of plant cells for electron microscopy. J Submicr Cytol 5:79-96. As I work exclusively with plant material, primarily leaves, it appeared to be best. The paper presents results indicatng that PIPES is the least extractive of buffers currently in use at the time. However, the work by Coetzee and van der Merwe (Coetzee J, CF van der Merwe 1987 Some characteristics of the buffer vehicle in glutaraldehyde-based fixatives. J Microsc 146:143-155.) indicates that the least extraction was found with phosphate-buffered glutaraldehyde. Again, their work was conducted using plant material (bean leaves) and may not be directly applicable to animal tissue, which was the subject of the original question. As most people know, phosphate is not compatible with added salts, hence, cacodylate would then be the buffer of choice. I would agree that morphologists are conservative in their approach to new techniques. I was extremely sceptical of the use of microwaves in fixations, until I was able to use one of the units myself. I am now convinced that this is a very powerful technique and yields fixation images equal to or superior to conventional protocols. The time-saving aspect is a tremendous bonus! I was loaned a Pelco unit by Phillip Slakmon of Scott Scientific for a number of months and have used it for more than a dozen fixations, which included standard protocols for both morphological and immunocytochemical studies. I have polymerized Spurr's resin, Epon-Araldite, LR White, and mixtures of Spurr and Epon, all with success (good infiltration, polymerization, sectioning, staining, and stability in the beam as criteria). Note that the polymerizations were conducted under water (!) in BEEM capsules. Has to be tried to be believed, I guess.

Anyway, I would hope that Partha would add his comments to this discussion on buffers, particularly regarding the suitability of PIPES for morphological work.

Dwight U. Beebe
E-mail: beebed@ere.umontreal.ca

Microscopists
With regard to all the dicussion on fixatives, buffers,etc; maybe it's time to suggest two references:

1.Coetzee,J.and van der Merwe, C.F.,Some characteristics of the buffer vehicle in gluteraldehyde-based fixatives.Journal of Microscopy,Vol 146 Pt2, May 1987,pp143-155.

2.Coetzee,J. and van der Merwe, C.F.,The influence of processing protocol on the ultrastructure of bean leave cells.S.African Journal of Botany,1986 52(2)

Alan Hall
HALL@scientia.up.ac.za

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