Frozen tissue fixation and sucrose immersion experiments

Summary

Tissue morphology is usually good for frozen sections if the tissue is fixed and macerated with sucrose prior to sectioning. Source: Practical Laboratory Techniques in Neurobiology

Operation method

basic program

Materials and Instruments

Frozen tissue
PBS PFA Sucrose

Move

1. Fix small tissue samples or isolated embryos (e.g. day 7 or 8 mouse embryos) in 2% PFA fixative (for immunohistochemistry) or 4% PFA fixative (for in situ hybridization) for 30 min at 4°C.

2. Wash the tissue samples twice in PBS. 3. Dip the sample in 30% sucrose until the tissue sinks (1-3 h to overnight).

Caveat

1. Large embryos and some tissues will need to be fixed for up to 4 h. Large organs should be fixed in situ by perfusion in the animal before being dissected down by the animal body.2 Frozen sections, the most common problem is ice crystals, to prevent ice crystals, you can tissue quick-freeze or use different concentrations of sucrose to replace the water of the tissue block, usually with 20% sucrose, can also be used to replace the 20%-25%-30% sucrose gradient, in addition to the configuration of the sucrose solution can not be prepared with distilled water, otherwise it can not play a dehydrating role. Tissue block by the sucrose solution can precipitate a lot of water, so the liquid should be replaced until the tissue block sinks to the bottom!

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