Be ready to decalcify for at least a week (not 48 hrs), with changing EDTA solution.

Hi Rustem,

The 4% PFA is usually diluted in PBS 1x.

For the microscopy, or any histology, I always fix the samples in 4% PFA for 24h at +4 degrees, and then we do the EDTA treatment to decalcify and then the embedding. I didn't try the formaldehyde / glutaraldehyde mixture. I think the fixation is really important and we always used 4% PFA in my lab in the USA.

Delphine

Bone should be fixed immediately after sacrificing of an animal, pieces of bone should be small (

Hi Rustem,

I work with tissue-engineered bones. I assume that's a little bit different than a native bone. But for TEM we directly fix our tissues in 2.7% glut at 4°C for 4 hours (depending of the size of your sample), then we wash them with 0.1 M of cacodylate buffer before a 30 min post-fixation in 1% osmium tetroxide. We obtained really good TEM pictures. Moreover, we observed that the time of fixation gonna increase the rigidity of your tissue and impact the quality of your cutting and finally the quality of your TEM pictures.

I hope the infos could help you.

Delphine, Vladimir and Fabien!

Thank you very much for your answers!

Now I am sure that the fixation does not interfere decalcification.

Since I do not plan on doing immunolabeling, we will fix the bone directly in a mixture of 4% formaldehyde (for quick penetration and stopping of all processes in the living cells) and 1% glutaraldehyde (for high-quality fixation for TEM) in 0.1 M cacodylate.

So protocol will be this:

1. fixation of 48 hours (or more) 4% formaldehyde and 1% glutaraldehyde

in 0.1 M cacodylate

2. Washing with 0.1 M cacodylate 3 times (or more)

3. EDTA decalcification (177 g / L in 0.1 M cacodylate) 48 hours or more

4. Washing with 0.1 M cacodylate 3 times (or more)

5. Postfixation with osmium tetroxide 2% in 0.1 M cacodylate

6. Washing with 0.1 M cacodylate 3 times (or more)

7 - Dehydration and inclusion in EPON with standard protocol.

Best wishes,

Rustem

Be ready to decalcify for at least a week (not 48 hrs), with changing EDTA solution.

Dear Vladimir !

Thank you for additional comments. We will use the cross-sections of bone taken with a high-speed rotary tool (Dremel 300, Dremel, USA) 0,4 mm thickness. This is not my first work on this subject. Just before the samples were fixed by Delphine, and now it will be done by other people. I wanted only to be sure in optimal protocol, because it will be the big experiment. For such small pieces at a volume of 1.5 ml of EDTA solution did not required changing last time.

What do you think  about the molarity of cacodylate buffer for fixation and washing - 0.1 M or 0.2 M is better?

Can I used water solution of EDTA or cacodylate is better?

Best regards,

Rustem

I am not sure what is better: after fixation water should be OK, but to be on safer side I use 0.1 M buffer.

Rustem:

I agree with Vladimir, you may be in EDTA for a week.  I used to do rodent lower jaws and we would keep them in the refrigerator until soft changing the solution every other day. Yes and keep the cacodylate to 0.1M.  Here is a protocol for comparison:

Decalcification/Fixation Protocol:

Fix samples in 2% parpaformaldehyde (freshly made ) + 2 % glutaraldehyde (both em grade) + 0.1 M sodium cacodylate  pH 7.2 for 1-2 hrs (1 hr/mm3 sample size) at R.T. or overnight 4 C.  Make sure of 10:1 fix vol. to sample size.

Rinse in 3 x 10 min 0.1M sodium cacodylate buffer pH 7.2

 Decalcify in 2.5-3 % (0.1M) EDTA + 2.5% glutaraldehyde + 0.1M sodium cacodylate buffer pH 7.2  (2 hrs/1.25 mm sample, or overnight-1 week 4 C).  Change solution daily. 

 3 x 10 min 0.1M sodium cacodylate buffer pH 7.2  

 1% OsO4 + 0.8% KFeCn6 (postassium ferrocyanide) + 0.1M NaCaco  1 hr dark on ice

 2 x 5 min D-H20 rinse ( RT-throughout )

 0.15% Tannic acid in D-H20 for 1 min-if you want cytoskeleton contrast-5 min for tissue

 2 x 5 min D-H20 rinse

 1 hr in dark 2% Uranyl Acetate (aqueous) 0.22 um filtered

 50% 70% 90% ETOH  5 min each

 3 x 5 min 100% ETOH (freshly opened pint sized bottle)

 2 x 5 min propylene oxide-do not let tissue be exposed to air surface for

any length of time-leave fluid on top of sample during exchanges

 1:1 propylene oxide: Eponate 12 (1:1 A:B) no catalyst capped, rotate overnight

 Next day 3 changes fresh 100% Epon (1:1 A:B)  with 1.5% DMP-30 catalyst.  You can include a 15 psi vacuum step (1-2 hrs) to better infiltrate hard to infiltrate samples.

 Cure in rubber molds or beem capsules (size 00) at 60 C for 2 days.

While reading the post& all it's replies (most of which I would second) I wonder (as I have done this not only in this special case) if there is a substantial reason for using "2.7% GA (glut)" for fixation of your preps, Fabian?

Thank you all!