We usually fix 16-20hrs in NBF, then do 5M EDTA pH=8.0 for 5-10 days at RT
until the bone has soften. Place enough EDTA and change EDTA once in a while
(like every other day) to avoid saturation of EDTA with Ca. After
decalcificaiton is completed I re-fix the bone is NBF for another 6-8 hours.
Then I transfer to 70% Ethanol for several hours and process for paraffin
embedding. We use longer program that yours:
Note: Make sure you use enough fixative and EDTA (15-20 times more than the
volume of the tissue itself) . Make sure the tissue floats freely in the
solution. You may even gently rotate while fixing. Make sure you wash with
copious amount of tap water between and after each NBF or EDTA treatment.
You can use harsher decal solutions for shorter times. EDTA is better to
preserve the target macromolecules If you do IHC, IF or ISH.
But your paraffin processing protocol seems short for that big bone you
have.
*Reagent *
*Station*
*Time (Hr:Min)*
*Temp °C*
*P / V*
*Drain Secs*
*Stir*
Ethanol 95%
1
2:00
AMB
AMB
120
On
Ethanol 95%
2
0:30
AMB
AMB
120
On
Abs Ethanol
3
0:30
AMB
AMB
120
On
Abs Ethanol
4
0:30
AMB
AMB
120
On
Abs Ethanol
5
0:30
AMB
AMB
120
On
Histoclear or xylene
6
0:45
AMB
AMB
120
On
Histoclear or xylene
7
0:45
AMB
AMB
120
On
Histoclear or xylene
8
1:00
AMB
AMB
120
On
9
10
Paraffin Wax
Left
1:30
59
P / V
120
On
Paraffin Wax
Middle
1:30
59
P / V
120
On
Paraffin Wax
Right
1:30
59
P / V
120
On
Good luck!
Mesru Turkekul
turke...@mskcc.org
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