Dear all,
I'm beginner of immunohistochemistry from dentate gyrus of mouse brain.
As you seeing from the attachment, my brain sections seem to be damaged.
1. many holes
2. especially, tear of subgranular zone(detachment between hilus and granule cell layer)
There is a difference in degree, but almost all the sections have the same problem.
What is the possible reason the sections were damaged like this?
Please feel free to share your opinions or experiences.
I'm looking forward to your reply.
Thank you.
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+ Following is the sample preparation protocol that I did.
1. perfusion by cold saline (did not the following perfusion of 4%PFA).
2. 48hr post-fixation by 4%PFA at 4℃.
3. cryoprotection by 30% sucrose at 4℃ until brains are totally sunk.
4. snap freezing by liquid nitrogen.
5. making OCT mold in dry ice.
6. cryosection(40㎛) at -21℃.(samples were acclimated in cryostat 30min before sectioning)
7.put all the sections in 24-wells plate filled with 1xPBS.
8. mounting on the slide.
Hi,
try adding crioprotection (sucrose) to PFA ant to make the fix step shorter eventually reducing you sample size (o.n. should be ok). Keep sucrose in postfix washing too.
Good luck,
Mariarosa
Hi Hwang,
Following few suggestions can help you...
1. Cryosectioning may have such drawbacks, such as it may leads to the formation of ice crystal in the tissue, results in the formation of such type of tearing and holes.
2. Gently remove the brain and try not to press it anyhow and as well as immediately transfer it to the formalin solution after washing in PBS. Pressure and oxygen exposure also results in such abnormalities in histology.
3. If you have microtome facility in your lab try sectioning on microtome of paraffin fixed tissue. you can cut thin sections as well on microtome.
4. To perform IHC of microtome sections follow protocol of Abcam attached here https://www.abcam.com/protocols/immunostaining-paraffin-frozen-free-floating-protocol#Paraffin%20and%20frozen
All the best!!! and you also feel free to discus further
You are clearly having tissue damage by the freezing. If possible use paraffin sectioning for such gentle tissue as the brain unless it is incompatible with the target that you are studying i.e. lipids. Another thing that you should drop is snap freezing in liquid nitrogen, slow freezing is better for tissue. I would suggest putting fixed tissue into the OCT molds put the molds into the styrofoam and in -80 so the samples freeze over the course of the few hours.
few good suggestions here, i use these techniques since 1997.. 1st, there might be some micro air bubbles when you mount thin frozen sections, when section comes off the blade put a finger under the section (under the glass slide) to help straighten the section. 3rd, as one person said, the damage may be during freezing. In your protocol you say "snap-freeze in liquid nitrogen". As another user said, slower (slightly slower) may be helpful BUT not by putting in -80 (these are not cells). What worked for me repeatedly is: either use isopentane dry ice bath (google it but in short - put isopentane in a jar (thermos, small styrofoam box), drop some 20 chunks of dry ice there, drop brains after 30% sucrose. When they are frozen, keep at -80. when you are ready to mount on a chuck and cut, put a drop of OCT on a cold chuck, let it freeze (30 sec), then put another large drop, stick your (well, mouse) frozen brain to a chuck using this freezing OCT as a glue, and then put more OCT over again and again to grow OCT coating. Trim excess of OCT with razor blade. Then cut. DO NT freeze already frozen brain in wet OCT on dry ice. This is probably where the problem happens (freezing-then partially thawing -and then freezing too quickly). another approach is: have ice bucket/stytofoam box 1/2 filled with dry ice (to have cold mist hanging over the surace), put a piece of foil on dry ice (kind of push foil into dry ice to create enough room for one mouse brain), wait ~5 min until you see the foil covered with frost (becomes slightly white -this means the area where you will put a brain reached the temp of dry ice), put mouse brian (after 30% sucrose) directly on the foil, and surround with more chunks of dry ice. You will be feezing at -55C rather than liq N2. This produces very high quality results: Article Long-Term, Stable Differentiation Of Human Embryonic Stem Ce...
also, when you section, you can keep sections in the wells of 48-well or 24-well or 12-well plate in the mix of Glycerol, ethylene glycol and 10x PBS, diluted to 1x (antifreeze medium). Then do staining of floating sections, then mount after 2ndary AB (DAB or fluorescent, does not matter). This works great. Last, when you stain frozen PFA-fixed brain sections with fluorescent ABs, dont be afraid to do dehydration as if you do DAB, it works and produces amazing results with high clarity because sections shrink (become thinner). do not switch to paraffin, 1st you are new to this and you dont need more challenges, histology is a challenging technique to master but worth it. 2nd, it also it also produces minibubbles in tissue. Good luck
Igor O. Nasonkin Thank you so much for your kind and helpful answer. I'm going to try to do IHC as your suggestion.
But I want to be received 2 more your suggestions.
1. would you let me know the detailed protocol of making antifreeze medium or any ready-to-use products?
2. you mean, mount brain sections on slide glass after completing 2ndary AB step by free-floating method and then do DAB or fluorescent steps by the mounted section on slides.
I'm looking forward your reply. Thank you.
indeed, store cryosections (individually, in 96-wells, which is hard to do due to the number of sections, or in bunches of 5 or 10 sections/well, in cryoprotectant solution consisting of EG (ethylene glycol), i believe 30%, glycerol (i believe 30%) , 10x- PBS (10%) and water, to make the final solution 1x PBs, with cryoprotectants. For those who work in neuroscience and routinely section brain/spinal cord, this is a routine approach. But if you were never exposed to this technique, this is an eye-opener. Cryoprotectant solution is described online, just google it, i believe i correctly described the cryoprotectant i used but there are several recipes, e.g. http://www.ihcworld.com/_protocols/histology/cryoprotectant.htm. The main point is that you never freeze the sections. For as long as a section is not falling apart you can do it (e.g., some cuts of the brain may give you few pieces, which are held by OCT but dissolve in cryoprotectant, leaving pieces of a section free floating, which is not good). Google free-floating IHC protocols, you will find many, i attached one
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