fixation of tissues using 4% paraformaldehyde

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kachang
kachang's picture
fixation of tissues using 4% paraformaldehyde

I am fixing rat ovary using 4% paraformaldehye for IHC.tissues are not intact but nick and crack appears around the tissues  on sectioning.what exact steps and timings should i follow.pls help

pundir
pundir's picture
 Hi,

 Hi,
the fixation time depends on thickness of your tissue, for rat ovary overnight would be enough also check the pH of PFA , clarify how you are processing your tissue after fixation means are you going for cryosectioning or paraffin sectioning if cryo then are you going for cryoprotection in 30 % sucrose befoere sectioning.............elaborate your querry for further help

kachang
kachang's picture
thank u pundir.

thank u pundir.
the ovary size is about 3cm x 3cm, PFA of pH7.4, and tissue processed in paraffin.tissue fixed for 72 hours as tissue doesnt get fixed in 24 or 48 hrs.fixed tissue are processed as follows:
PBS 1x wash - 3x -30 min each
dehydration in alcohol series of
30%,50% -30 min each,
70% -overnight,
90%,100% -30 min each,
100%:xylene-20min
xylene I & II -20 min each
Xylene : wax - 45 min
wax I & II - 45 min each
wax III - 1 hr
embedding

drgaju.15
drgaju.15's picture
hai

hai
can any body tell me 4% paraformaldehyde and 10% formalin is same or different

thanks

Matizales
Matizales's picture
Hello,

Hello,

I hope you solve your doubts.
Anyway, 4% formalin and 10% formalin is the same, as it comes from 35-37'5% and you could consider it as 4% (37%) or 10% if you add 9 times solvent.
Paraformaldehyde is the polymer and you can consider it in the same way. You will use PFA when the fixation concentration is critical.

Good luck,

Pedro