Hi Samad!

When GA is exposed to air than oxidation starts. The aldehyde turns (very) slowly into Glutaracid. This acid could destroy the ultrastructure of your samples. So I would suggest not to store your samples in GA over a long period (1 month). After fixation I put the samples in Cacodylatbuffer (0,1 M).

Best,

Eric

as far as the brain is concerned, there is no way you can overfix samples for electron microscopy. so I guess your  samples can stay in glutaraldehyede quite long.

In my experience: fix samples (30 um slices cut with a vibratome or little pieces of tissue) in:  2% glutaraldehyede in 0,1% Na cacodylate buffer pH 7,2 + 5% sucrose for 45-60 min at 4°C. Remember to cut the sample into smaller pieces if needed, after 10 min of fix. For samples with a thickness of 2-3 mm, the time to optimal fixation in glutaraldehyde is about 2 hours, always at 4 °C up to a maximum tolerated 12 hours. Do not exceed the recommended fixation times because samples tend to harden and antigens may be masked and no longer recognizable by antibodies (if you want to perform immunostaining with colloidal gold).

Wash well for at least 1 hour in buffer + 5% sucrose (or overnight at 4°C). Glutaraldehyde fixed well the protein structures but to preserve the lipid structures and in particular the membranes, it is necessary to perform a second fixing, or post-fixation, using a solution of osmium tetroxide. So, post-fix with 1% OsO4 in cacodylate buffer 0,1M pH 7,5 for 60 min at 4°C, wash well (at least 20 min) and proceed with dehydration and embedding.

Good luck and let us know

Barbara

Hi Samad!

When GA is exposed to air than oxidation starts. The aldehyde turns (very) slowly into Glutaracid. This acid could destroy the ultrastructure of your samples. So I would suggest not to store your samples in GA over a long period (1 month). After fixation I put the samples in Cacodylatbuffer (0,1 M).

Best,

Eric

Dear Samad,

Hi again, How are you?

As you know, preparation of Specimens for transmission microscopy should be follow up as below stages:

1] Fixation Perfusion or immersion fixation of small blocks

2] Fixatives – Aldehydes (glutaraldehyde); Osmium tetroxide

3] Dehydration

4] Embedding – orientation of the specimen Epoxy resins

5] Staining – Uranyl acetate; Lead

6] Cutting sections: 1 micron for light microscopy and ultrathin sections

7] How to get the best out of the microscope and the specimen good quality,

well fixed,

well orientated and embedded,

well cut and well stained

viewed in a microscope in good condition.

If you do not have the best quality you are wasting your samples and time!

Dear researchers,

Thanks a lot for your answers. I will consider all the explanations into account.

Best regards.

I am not a big fan of long term storage in fixatives, especially primary fixatives.  Remember the glutaraldehyde will not fix lipids, so with time these will be lost/extracted.  If we receive tissue on Fridays, the longest I would go in primary fix is over the weekend at 4C.  If possible it is better to get through the secondary osmium fixation, then store in buffer.  Although I understand that sometimes you cannot avoid long term fixation storage, hopefully you have a control to compare conditions with.

Good Luck.

Glutaraldehyde continues to make more bonds and crosslink so long as there is active dialdehyde, and a significant problem is hardening of the outside tissue surfaces preventing further penetration into the inner sample; it can also self-polymerize. It should be washed out after a couple of hours, or there is a risk that this crosslinkage (of proteins only) will compromise your ultrastructure. The treatment has stopped enzymatic hydrolysis, however, so fixation has served its main purpose.

Standard fixation procedures usually recommend only a couple of hours in aldehyde (you may find a formaldehyde-glutaraldehyde mix more effective for that type of tissue). Then there may be a slight delay before continuing to get the other components fixed (lipids by osmium tetroxide, or directly into dehydration/plastic for immuno procedures): but this delay shouldn't be long-term; get the tissue processed without sitting in buffers and such that can leach or otherwise compromise the tissue material. Those who do not fix attentively have inferior sample results.

You can keep in primary fixatives (Karnovsky's) for maximum 12 hrs (2mm thick section) and afterwards you can store in PBS (0.1M PB) in 4 degree temp for long.