[Histonet] Re: Histonet Digest, Vol 93, Issue 14

[mesruh turkekul]

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
turkekul <@t> mskcc.org