1. Fixation in 10% neutral Formaline at least for 24 hours (better 48)
2. Then there are several possible ways. If you are not running out of time - it is better to use EDTA-decalcification protocol (2a). If you have to make it fast - you should use acid decalcification (2b).
2a. So, EDTA decalcification will take app. 3 weeks. You will need to prepare 10% EDTA (pH 7.4) and change the solution every week.
EDTA 10% for 500 ml:
- 50 g EDTA disodium dihydrate
- ddwater 400 mL
- app. 3 ml 5M NaOH
Mix well and adjust pH to 7.4 with NaOH
Add water to 500 mL.
!!! The meterial/decalcifing agent ratio should be at least 1:20 !!! For EDTA it is recommended to have this ratio 1:40
2b. There are a lot of different acids for rapid decalcification. My personal preference is 9% formic acid (maybe not the fastest, but the tissue damage is also not so severe comparing to HCl and HNO3).
- 90% fromic acid - 10 ml
- ddwater - to 100 ml
(adjust the proportion according to the desired volumes)
For the specimens of your dimensions I think it will take 1 to 2 days. But start to check for end point after 24 hours.
3. Establishing decalcification end point. Well, the best way is to use X-ray. If it is unavailable - then you can just try to band the bone: if it is banding, then decalcification is ended. Or if your pieces are too small for bending - you can try to cut it: if you can cut it with surgical blade - then it is ready. Or you can use ammonium solutions for the chemical test.
Good luck!
Here is a well-written, detailed methodology:
http://grossing-technology.com/newsite/home/grossing-techniques/decalcification-in-surgical-pathology/
In short, if it is a small specimen (like a needle biopsy from bone marrow biopsy) it may be best to use a superficial decal (couple of hours, weak acid like formic acid or acetic acid) and if it is a large piece of bone, you will probably have to use a strong acid like 10% HCl, likely overnight if not more.
Keep in mind, decal just for H&E is fine, but for a lot of immunohistochemical stains, decal process will denature the epitope recognized by the antibody & may limit the interpretation of the stain. Good luck.
It is difficult to answer your question not knowing the size of your specimens and whether you intend to have histochemical stains only or try immunohistochemistry. I have attached the Leica Biosystems 'An Introduction to decalcification' http://www.leicabiosystems.com/pathologyleaders/an-introduction-to-decalcification/ which gives a good overview of different methods. This text complements in some respects the one send to you by Grzegorz.
I have used different acids depending on what I wanted to achieve. For diagnostic material from bone tumours we used formic acid for rapid decalcification. For samples which need immunohistochemistry I use EDTA. If you work on archival blocks with undecalcified bone which were cut on sledge microtome and need H&E histology only - surface decalcification works well. Good luck with your work.
I agree with Dr Gurda. When we plan to do an immunohistochemistry in a bone sample, we need to decalcify with a mild decalcifier solution, such as Osteosoft (Merck). It is much slower than the acids, but preservs epitopes for immunos.
Where simple histology is required we use immersion in 20% formic acid. Tissue is fixed first in formalin. Small core samples may decalcify overnight but large slab samples that need to be trimmed again could require several days and should be monitored daily. Once the sample size is appropriate for embedding rinse in running water for 20 min and then neutralize in 5% sodium sulfate for at least one hour. Rinse a second time for an hour to overnight and then embed in paraffin as usual. Inadequate washing and/or neutralization will result in poor H&E stain uptake.
Do you read answers to your question? Like Maria Jeziorska have already mentioned: if you need a precise protocol - tell us WHAT you want do decalcify (tissue type, size of the sample) and for what PURPOSES (general morphology, histochemistry, immunohistochemistry).
You can also use ultrasound, but it takes much longer.
http://www.ncbi.nlm.nih.gov/pubmed/16819333
1. Fixation in 10% neutral Formaline at least for 24 hours (better 48)
2. Then there are several possible ways. If you are not running out of time - it is better to use EDTA-decalcification protocol (2a). If you have to make it fast - you should use acid decalcification (2b).
2a. So, EDTA decalcification will take app. 3 weeks. You will need to prepare 10% EDTA (pH 7.4) and change the solution every week.
EDTA 10% for 500 ml:
- 50 g EDTA disodium dihydrate
- ddwater 400 mL
- app. 3 ml 5M NaOH
Mix well and adjust pH to 7.4 with NaOH
Add water to 500 mL.
!!! The meterial/decalcifing agent ratio should be at least 1:20 !!! For EDTA it is recommended to have this ratio 1:40
2b. There are a lot of different acids for rapid decalcification. My personal preference is 9% formic acid (maybe not the fastest, but the tissue damage is also not so severe comparing to HCl and HNO3).
- 90% fromic acid - 10 ml
- ddwater - to 100 ml
(adjust the proportion according to the desired volumes)
For the specimens of your dimensions I think it will take 1 to 2 days. But start to check for end point after 24 hours.
3. Establishing decalcification end point. Well, the best way is to use X-ray. If it is unavailable - then you can just try to band the bone: if it is banding, then decalcification is ended. Or if your pieces are too small for bending - you can try to cut it: if you can cut it with surgical blade - then it is ready. Or you can use ammonium solutions for the chemical test.
Good luck!
The method you use depends upon what you need to do with the sections following decalcification. If you want to use any molecular methods requiring intact DNA, then use the buffered EDTA described by Dr. Fedchenko. To decrease the time, you might also add a cationic exchange resin to the solution and agitate this twice or more per day. For immunohistochemistry and histology, 20% formic acid in 20% formalin with added cationic exchange resin will decalcify tissue 3-4 mm in thickness in one day at room temperature--and you don't have to prefix the tissue and you don't need to wash for as long afterward prior to fixation to make the tissue stain archivally. In addition, if you forget about the tissue for a few days the histology is not significantly harmed by this method (I've put a piece of bone in this solution for over a year and could still get readable sections). This method works for every immunohistochemical agent which I've tried, but it doesn't work for in situ hybridizations and I suspect it won't work for chromosomal probes though I haven't tried it with these.
What is the diiferent of decalcification result using Rapid Immuno and EDTA ?
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