I am trying to perform H&E staining on mouse guts (Swiss Roll). I have fixed the guts in NBF at room temperature overnight before preparing the Swiss Rolls. The cryosections that I made seem to be pretty good. However, when I started staining the tissue sections, they seem to be tearing off from the surface of the glass slide. I am using SuperFrost slides. Any suggestions on how I could prevent loss of tissue ?
Also, once I am done with the H&E staining, what is the best way/software to quantify the size of the polyps in the samples? Any help would be greatly appreciated.
Thanks
Hi
I suggest glycerin+chicken egg albumin mixture as glue for your section. Probably it solve your sample preparation!
I suggest zeiss-Axiovision software for your histomorphometry and size measurement of polyp. Good Luck
Hello,
We used economy microscope slides we cover them with gelatin. Regards
Hi Vidya!
Have you tried fixing the tissue prior to staining. incubating cryosection in 100% acetone at -80 degree for 15 min would help in the problem you are facing.
good luck
Concerning the quantification of polips I would suggest Image J as the first choice. Best wishes!
Hi
I use gelatin coated slides for my swiss roll sections and it works a treat. I use the protocol given on R&D systems website.
Prepare the gelatin-coating solution by dissolving 5 g of gelatin in 1 L of heated, deionized H2O (temperature should not exceed 45 °C).
After the gelatin has dissolved, add 0.5 g of chromium potassium sulfate dodecahydrate. Chromium potassium sulfate dodecahydrate will positively charge the slides allowing them to attract negatively charged tissue sections.
Filter this solution and store at 2-8 °C until use. It is recommended that this solution be filtered again immediately before use (adjust to room temperature before filtration).
Place the histological slides into metal racks.
Note: The slides should be cleaned by washing them in soapy water and rinsing them thoroughly, first in tap water and finally in deionized water.
Dip the racks containing the slides 3 to 5 times (~5 seconds each) into the gelatin-coating solution.
Remove the racks containing the slides and let them drain. Blot excess solution from the racks onto filter paper (gently tap the racks against the filter paper for better drainage).
Place the racks containing the slides on the lab bench and cover them with paper towels to protect them from dust.
Dry at room temperature for 48 hours.
Dried slides can be put back into the boxes that they arrived in and stored at room temperature until use. Slides intended for cryostat sections can be stored at -20 °C.
You can also use APES solution.That is also good.
Best of luck,
Breedge
Hi vidya,
If you want to save time then u could use poly-L-Lysine coated slides. They are pre-coated slides supplied by thermo scientific, and I use these slide for my work as well.
As far as analysis of image is concrned Image J is good, but Volocity is even more better.
Hope this helps.
polly
I always used poly-L-lysine coated slides for cryostat sections of gut, brain, muscle, other tissues and never lost sections during immunostaining.
I recommend you to do use a dryer while sectioning and MAS-Coated slides works really nice.
good luck
I recommend you to do use a dryer while sectioning and MAS-Coated slides works really nice.
good luck
According to my practical experiences, you are recommended to use one drop poly-L-lysine (100 microliter poly-L-lysin + 900 microliter DEPC treated water) onto the slide and then dry at room temperature for around 10 minutes before attaching the section. You are also recommended to dry the section for at least 30 minutes by using air dryer in cold mode immediately after keep out from freezing temperature. All the best.
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