Dear colleagues I am looking for a freezing tissue protocol which can preserve good morphology and antigenicity for immunofluorescence. The scientific literature is scarce on this topic. I am not sure how to snap frozen, liquid nitrogen or isopentan? How to store samples, in -20, -80 or liquid nitrogen, which option is better? OCT cryopreservative should be used to store samples? Can it improve morphology quality?
There are few ways: you can place tissue in a crymold, fill the cryomold with OCT. and place on dry ice or throw in LN2. Second method is Best way to preserve cell morphology is: take the tissue, fix in 4%PFA for 1 to 2 hours or overnight at 4C. Than rinse once in PBS, place in 15% sucrose, till the tissue settles down, than place in 30% sucrose, till it settles down. It may take 2 days in sugar step.
Than place tissue in the middle of a cryomold, fill with OCT, and place this cryomold on a horizontal piece of dry ice, while you can orient the tissue immediatley as it starts freezing. after 30 minutes, everything frozen, store the frozen cryomold with tissue in a box at -70, -80, freezer whatever is available. No need of liquid nitrogen here. Once you made cryosections, bring cryomold to -22C in crystat and cut them. After you got sections, you may fix again in 4%PFA for 10 minutes and than proceed with your immuno. If you have any question, please let me know
Alternatively, cryosections you may fix in glutaraldehyde, here is procedure in the paper attached, hope this helps you,
I never recommend snap freeze by liquid nitrogen unless required by specific staining. I usually use the following approach, mild tissue fixation with 4% PFA or Histolab fixative for 2 hours at room temperature or overnight in 2% PFA at 4C.
In your case, the length of fixation entirely depends on the skin sample size. If you are working with biopsy disks 2 hours at RT in PFA4% should usually be sufficient.
Another approach my colleague use with good success with cornea and skin is incubation in -20 acetone, compared to PFA it improves the tissue immunogenicity at the expense of the slightly less favorable morphology and complete loss of any lipids in the tissue. For embedding first place tissues in 15% sucrose in PBS until tissue sinks (6-12 hrs) and then 30% sucrose in PBS for overnight or until tissue sinks to the bottom of the tube. Embed the tissue in OCT and put the molds inside the styrofoam container in -80 so the freezing occurs slowly. Then you can proceed with sectioning. The skin is a fairly hard tissue and easy to produce good quality histology. I usually store OCT blocks in -80 in a sealed bag to prevent drying of OCT. Sections can be stored at 4C.
Thank you very much for sharing these information. Is it necessary to perform antigen retrieval if I fix with 4%PFA for two hours? Anton Lennikov Subhash C. Juneja
In frozen sections, generally antigen retrieval is not required. I always use antigen retrieval in paraffin-embedded sections
Hi Anton Lennikov
Do you mind post one publication you used with the acetone protocol. I got very interested in try it.
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publication