I want to cut 25μm longitude section of mouse gastrocnemius muscle in order to stain the NMJ with synaptophysin and alpha-Bungarotoxin conjugated with Alex 594.
Here is the frozen section protocol I used: I dissect out the gastrocnemius muscle from mouse, rinse it with PBS, and then embed into a thin layer of O.C.T onto wood cork; prechilled the 2-methlbutane (in a glass beaker) in liquid nitrogen till the white precipitate starts to form at the bottom, then immerse my specimen completely in 2-methlbutane for 45 seconds, immediately lay my frozen specimen onto dry ice for about 15 mins; I store the specimen at -80c overnight, and put it into -20c cryostats 30 mins before sectioning.
However, it is really hard to cut complete and smooth 25μm longitude section without getting holes and crimping, even though the blade is brand new. Still, I did the IHC with synaptophysin, BTX and DAPI mounting media. only little area looks normal, most of the nucleus are not round shape but more like being dragged and twisted.
Is the problem of water crystalization or long freezing time? Which step can I make changes to avoid this improper freezing?
PS. I use snap freezing instead of 4% PFA fixation because the later one requires antigen retrieval protocol for my synaptophysin antibody.
Dear Ms Yang: Longitudinal cryostat sections of skeletal muscle picked up with glass slides onto which a film of ethylenediaminetetracetic acid (EDTA) has been dried after dipping in a 3% EDTA solution, do not show the severe retraction artifacts Histochemical techniques work well on the longitudinal sections after the EDTA has been rinsed away. Morphologic preservation is good. Tateki Kikuchi
Thank you very much Dr. Kikuchi! Should I rinse away the EDTA by PBS buffer before blocking step in IHC?
I tend to leave my tissue in the cryostat for at least an hour prior to sectioning - the longer to equilibrate, the better.
And I agree that the samples should be rinsed with PBS prior to blocking. Best of luck!
Dear ms Young... we put all muscles that we do frozen sections on, including gastrocnemius, on aluminium foil, put the foil direct in liquid nitrogen and wait that the muscle freezes. It is very important to keep the muscle dry before freezing with liquid nitrogen...any excess PBS or other liquid makes big tissue artifacts (tearing and holes) so maybe your problem is becouse you rinse with PBS.
To evaluate whether this is water crystallization, stain a H&E. You can clearly identify normal and water crystallization easily (see attached images).
If water crystallization is evidenced, you may want to add two step before OCT embedding: 1. put samples in 15% sucrose in 1X PBS for several hours; 2. transfer samples to 30% sucrose in 1X PBS for several hours. This will help remove intercellular water.
Hi, you can't use sucrose if the sample is not fixed first. The best freezing is done with iso-pentane cooled to just before freezing in liquid nitrogen and the sample is just dropped into the iso-pentane. Sample should be stored in liquid nitrogen or -80C if needed and let to equilibrate in the cryostat for sometime before sectioning. Also check that the cryostat is the correct temperature.
PBS-treament / rinsing of the specimens directly prior to "embedding" the then wet muscle into "into a thin layer of O.C.T"
most probably produces an "isolating layer" disturbing rapid and thorough/equalyy/evenly cooling of the whole specimen.
Insofar, I second 100% the argument of Mayda Maasarani [Re #04] above: "It is very important to keep the muscle dry before freezing with [NB: precooled isopentane nearly at its solidification point by incubating in] liquid nitrogen...any excess PBS or other liquid makes big tissue artifacts (tearing and holes) so maybe your problem is becouse you rinse with PBS."
and the comment {#07] by Mary Simonian: "The best freezing is done with iso-pentane cooled to just before freezing in liquid nitrogen and the sample is just dropped into the iso-pentane. Sample should be stored in liquid nitrogen or -80C if needed and let to equilibrate in the cryostat for sometime before sectioning. Also check that the cryostat is the correct temperature."
Dear Ms Yang,
I agree with Mayda and Wolfgangs statement - and most of the OCT users never looked at the ultrastructural level of what OCT does to the structure - even if you have very little of "water" itself binds water and if you freeze it the "tissue becomes" soft frozen ready for semi-thin cryo-sections but at the level an electron microscope can see (and also a trained eye of a pathologist in LM by the way) - OCT embedded tissue still show segregation pattern from ice crystal formation - see two images of OCT infiltrated and carefully embedded OCT human skin tissue - the empty areas in the tissue and in the OCT pure glue are areas were ice crystal have been formed and were that water was removed by freeze drying to show where the ice crystal formed in a tissue. The more water you have (PBS solution or other buffers) the larger these water crystal area get and hence the more difficult the sample is to be sectioned - it can even become brittle and not only show distortion. This is the reason why you should dry the sample from any excess water by exchanging it into fresh OCT a few times or embedded as Xjaojian proposed bind free water by using high concentration of cryo-protectant (sucrose see Tokuyasu method from EM methods, dextran 20% etc..).
The best is to exchange OCT a few times before you freeze the sample and try the freezing slowly on Al-foil and try sectioning again if you do not like to use sucrose, dextran and PFA etc..
Good luck and hope the "ice segregation pattern" in the attached image gives you an idea that OCT is not a perfect cryo-protectant at all.
Roger
Dear Roger, perfect! Thanks a lot for posting as well as providing SEM-micrographs of FD OCT-"embedded" muscle /skin specimens.... Hope you are fine, have a great Winter time in Australia....Best wishes and regards, W.M.
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Histological Techniques