I'm looking for paraffin embedding protocols to use on blastocyst stage embryos in sheep. If anyone has a protocol or some reference, I would be very grateful. Thank you.
Dear Federica,
This protocol for murine blastocysts, I hope it will be useful for you
http://www.rbmojournal.com/article/S1472-6483(10)61804-4/pdf
(Paraffin-embedded manipulated blastocysts)
Regards,
Islam
Hi Federica,
Just curious, what is the approx diameter of sheep blastocyst.
Subhash
Hi Federica,
If I were you, I will go for frozen sections rather than paraffin sections. I feel frozen sections will be more convenient than paraffin section. You will go thru less steps and less fear of losing embryos.
A. Place all the embryos in a 25ul drop of medium on a 60mm-dish under dissecting/stereomicroscope
B. Add 25ul 8% PFA (final will become 4%). Wait 5 min, remove this solution (under microscope). Add 100ul PBS with 2%BSA, 2 times for washing.
C. Place 100-200ul OCT drop in the middle of histological cassette
D. Transfer fixed embryos in (in step B) in a minimum volume of PBS (with 2%BSA) and mix gently onto OCT drop (on the cassette) (try this step under microscope, but not necessary) so that PBS gets diluted and OCT takes over. It is better embryos are suspended in the drop (which will be, since OCT is viscous) and not touching the bottom of cassette (you may have room for trimming while sectioning)
E. Place the cassette on a slab of dry ice, and immediately pour more OCT around that drop, Leave there for 30 minutes to freeze
Now your embryos are in the middle of cassette with OCT, and you can cut via Cryostat or store block at -86C or in LN2
Advantage of frozen sections will be that you do not need antigen retrieval step for immuno-histochemistry,
You can modify tiny steps of this protocol as you wish,
That is what I can think of,
Good luck,
Subhash
Hi Federica,
I forgot to add one more step, after step B, after removing PBS, place a drop of 15% sucrose for 15 min and than 30% sucrose for 30 min, than continue with step C (transfer in a minimum volume of 30% sucrose in OCT drop on cassette),
SJ
In routine procedure with glicol-metacrylate for obtaining of thin histological sections.
Hello everyone,
I would like to ask you if any of you has an recommendation for preservation method for uterine horns of E6.5 embryos. I want to get sections of embryos surrounded by extraembryonic tissue and within the uterus. The sections will be stained with an antibody for immunohistochemistry. People suggested to me 4% PFA O/N at 4 degrees and then parrafin embedding but with a two-step protocol (first paraffin inside the embryos and then embedding of the ''parrafinized'' tissue in parrafin. However, I dont know how to achieve the insertion of paraffin inside the embryos (which will be inside the uterine horn). Any help/advice would be much appreciated!
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