Hi everyone,
I am preparing cryosectioned slides from mouse E9.5 embryos but I am facing several difficulties. First, embryos at this stage are too tiny and fragile, it is really difficult to transfer a whole embryo to a cryomold without distorting the embryo. Secondly, I do not know who to remove PBS solution completely before adding OCT compound to the cryomold. I was wondering if there are som special techniques or tricks to make nice cryosection from E9.5 embryos . I am looking forward to hearing your experience and advice. Thank you very much!
I understand that you facing problem in handling these embryo. Once you take out embryo in dish, leave the embryo/s in the same dish, remove everything, add 4% PFA and close the dish with the lid and leave in refrigerator for 1 hour so that embryo/s get fixed. Ideally, you can replace 4%PFA with 15% sugar and than 30% sugar overnight. Sugar steps you can skip if you want.
You have pipettor and 1 ml tip in the lab. Cut the tip at the end with a blade, make a wider hole. Gently pick up (very slowly) embryo suck in the tip with the help of the pipettor.
Alternatively You can use glass pasteur pipette and use rubber bulb to suck and cut the hole to wider hole with a diamond pensil and handle that way too.
Try plastic 'transfer pipette'.
either way, You can place the embryo in cryo mold (no PBS in that), orient the embryo under dissecting microscope. Place cryomold with embryo on dry ice and add immediately OCT in the mold. Dry ice block should be horizontal. you need a small piece.
Hope that helps
I deal with embryos in such stages, I can give you some points from my experience:
1. First, you need a set of microsurgical instruments to dissect and collect the embryos.
2. Then, use a stereoscope to visualize while handling, dissecting and transfering the E9.5 embryos.
3. For transfering the embryo to the mold, use the perforated spoon (a microsurgical instrument).
4. I also prefer to use eppindorf tube 1.5 ml to incubate the E9.5 embryos in PFA and PBS solutions.
5. I advice you to use 1% of PFA instead of 4%, since embryos at this stage are VERY delicate, and also it gives better immunostaining results if you plan to do so.
Voilà
GOOD LUCK
Subhash C. Juneja, Hasan A Asfour : Thank you very much for your sharing!
I have tried to fix embryos in 2% freshly made PFA for 20 - 30 min, then transfer them to 10%, 20% and 30% sucrose solutions before embedding in OCT. Here, I faced two problems again. First, for some reason the embryos became flattened on the side which was in contact with the bottom of containers (12-well plates for example) after fixation step. Second, it seemed that 30% sucrose made the embryo more rigid so it was sometimes impossible to get a section of it.
As I am aiming for immunofluorescent staining only, I am thinking to skip PFA fixation step before embedding. A perforated spoon is something worth to try. Again, thank you for your suggestion!
That is ok, if you feel spoon works, that is good. Or try foreceps curved at the end. PFA or sucrose you can skip or both of them if you feel uncomfortable. I have also done directly thrown tissue in OCT and than place the cryomold on dry ice or -70 freezer. Still you should be able to do it. In this case, when you do cryosectioning, you fix the sections with a drop of PFA for 30 minutes on the slide, wash with PBS and proceed with immuno
Last step: take the slide with section out of freezer. Leave horizontal for 30 minutes at room temperature so that section settles at the bottom of slide (important step). Wash section once with PBS very gently to dilute OCT for 5 minutes, than place PFA and proceed
I drop fix in 4% PFA and as mentioned above use the transfer pipet method for moving the embryos around. They sell very cheap plastic eyedroppers (ie Fisherbrand Disposable Polyethylene Transfer Pipets Cat no. 13-711-9AM). Upon dissection, I place the E9.5 embryos into a single tube of cold 4%PFA, leave it on a rocker O/N in the cold room. The next day, I do about 3 rinses with cold PBS using a 1ml pipet and being careful to try and keep embryos towards the side of the tube so they don't get aspirated up during washes. I then dehydrate in 30% sucrose, again on rocker O/N 4C and then the next day when I go to embed, I actually use those disposable plastic spatulas to "scoop" out my embryos, place them into a disposable little weigh boat, and then use the spatulas again to put into the molds, which I have set up on metal blocks sitting on dry ice and embed.