I just would like to know a few general tips - What thickness should sections be? What temperature should the cryostat be? Does orientation of the tissue on the chuck matter? Would you recommend a particular lobe or area of the liver to use? Should I leave the sections to air dry once they're on the slide (How long? At room temperature or 37°C?)? Can the slides be stored for long once the sections are on them (how long? at what temperature?)?
I intend to perform H and E, as well as Oil Red O staining on the sections.
That's a lot of questions, I know! But any advice at all would be greatly appreciated.
Thank you.
Paul
- may want to consider freezing in pre-cooled isopentane, instead of liquid nitrogen; in isopentane it freezes virtually immediately, whereas there is a lag with liquid nitrogen (the nitrogen evaporates, creating a sheath of air around your sample). (Be careful, though, you isopentane evaporates, is toxic and extremely highly flammable). Fast freezing is important to avoid generation of crystals. You can also consider prefixing with 4% paraformaldeyde, and incubating sample 1-2 days in 30% sucrose; the sucrose inhibits crystall formation.
- regarding cutting temperature: I cut usually around -20C. Ideally, there should be a little bit of a difference between the knife, and the surface that receives the section. If your section melts, the surface is too warm, if it rolls up too fast to be able to collect it, its too cold. Rolling up happens often when you did not let the sample equilibrate to the cutting temperature (for instance, if it was stored at -80). I don't like cutting at very low temperatures (-30C), because the tissue tends to get brittle.
-lobe etc depends on what you want to see. Liver is composed of repetitive units, so if you are not interested in gallbladder or particular blood vessels, orientation should not matter too much.
- sections should not be thicker than 8 um (that is ~ 1 - 1.5 cell layers). Otherwise, in HE etc. it becomes too opaque. The thinner the better, but sometimes it can be hard to make very thin sections. If you do fluorescence staining, you can get away with thicker sections (10 - 15 um; for confocal even 20 - 30 um).
- slides can be stored at least for several weeks at -20C, and even for months at -80C.
- I think slides are okay if they sit at room temperature briefly (20 minutes or so) before being transfered to freezer.
- I air dry slides just before starting staining procedure for 5 - 10 minutes at room temperature.
- make sure to use coated slides (superfrost), so sections stick to your slide; otherwise they will detach during staining procedure.
- for HE may want to consider paraffin, its easier to get high quality sections, and the embedded sample can be stored for years.
Good luck
Hannah
HI Paul,
cut sections 5-15 μm thick in the cryostat at −20°C. Within 1 min of cutting a tissue section, transfer the section to a room temperature microscope slide by touching the slide to the tissue.The tissue section will melt onto the slide. You can store unused tissue in the freezer. For long-term storage, a moistened tissue should be added to the container with the block to prevent desiccation. For cryosections is best to use unfixed tissue, just make sure it's well frozen. I think the orientation shouldn't matter. good luck
- may want to consider freezing in pre-cooled isopentane, instead of liquid nitrogen; in isopentane it freezes virtually immediately, whereas there is a lag with liquid nitrogen (the nitrogen evaporates, creating a sheath of air around your sample). (Be careful, though, you isopentane evaporates, is toxic and extremely highly flammable). Fast freezing is important to avoid generation of crystals. You can also consider prefixing with 4% paraformaldeyde, and incubating sample 1-2 days in 30% sucrose; the sucrose inhibits crystall formation.
- regarding cutting temperature: I cut usually around -20C. Ideally, there should be a little bit of a difference between the knife, and the surface that receives the section. If your section melts, the surface is too warm, if it rolls up too fast to be able to collect it, its too cold. Rolling up happens often when you did not let the sample equilibrate to the cutting temperature (for instance, if it was stored at -80). I don't like cutting at very low temperatures (-30C), because the tissue tends to get brittle.
-lobe etc depends on what you want to see. Liver is composed of repetitive units, so if you are not interested in gallbladder or particular blood vessels, orientation should not matter too much.
- sections should not be thicker than 8 um (that is ~ 1 - 1.5 cell layers). Otherwise, in HE etc. it becomes too opaque. The thinner the better, but sometimes it can be hard to make very thin sections. If you do fluorescence staining, you can get away with thicker sections (10 - 15 um; for confocal even 20 - 30 um).
- slides can be stored at least for several weeks at -20C, and even for months at -80C.
- I think slides are okay if they sit at room temperature briefly (20 minutes or so) before being transfered to freezer.
- I air dry slides just before starting staining procedure for 5 - 10 minutes at room temperature.
- make sure to use coated slides (superfrost), so sections stick to your slide; otherwise they will detach during staining procedure.
- for HE may want to consider paraffin, its easier to get high quality sections, and the embedded sample can be stored for years.
Good luck
Hannah
Totally agree with Hannah. I think you should consider paraffin for HE as it will guarantee an intact morphology of your tissue, which is not the case with cryosections.
Concerning the hepatic lobe to study, no matter but once you chose the lobe, be sure to always work on the same lobe for all your studies. May be you should first characterize your model to confirm homogeneity of the pathology/phenotype between the hepatic lobes.
Enjoy
Benoit
Just one remark for cryocutting. I do fixation immediately after cutting, meaning that I attach the section and put it in 95% v/v ethanol solution. It stays there for 15 min and afterwards is rinsed in distilled or tap water.
Cheers,
Ivan
hello: i agree with many of my colleagues but I think paraffin sections will let you control the situation better and you get good results when you stain either H&E or even Histochemical stains. you obtain a good morphology and preserve the slides for further examination.
one thing about research is many times i have gone back to my old slides and saw items i missed the first time.'
i know croysections are faster and quick but if you intend on through examination i feel paraffin sections are better. As for orientation yes it is very important to orient to the direction of the lobe you want to examine ,i usually draw on a piece of paper the orientation of the liver and the lobe and keep it in the file believe me if you don't have orientation many lobes look alike and one tends to blame himself by saying i wish i know which lobe is this??
I use professor john kiernan’s book on histochemistry he is also my colleague in my department and a well respected histologist and histochemist in the world
You can see all his achievements here http://publish.uwo.ca/~jkiernan/aboutjk.htm and his new book
Kiernan, J. A. 2015. Histological and Histochemical Methods: Theory and Practice. 5th edition. Banbury, UK: Scion Publishing. (ISBN 9781907904325). has just been published and is available
good luck and best wishes
Professor K.Galil.DDS.,D.Oral&max fac Surg.,Ph.D.,FAGD.,FADI.,Cert.Periodontist (Royal College of Dental Surgeons)
Patenet developer of website , visit to see the new Oral histology App for the I phone,Ipad &I Pod touchhttps://itunes.apple.com/us/app/dgohe+/id1015383607?mt=8
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