HISTOTECHNOLOGY
- 10% formalin and 4% paraformaldehyde are the same thing. Store at room temp. Used for paraffin-embedding protocols.
- 10% formalin/ 4% paraformaldehyde + ethylene glycol and sucrose solution. Store in freezer between 4C and -20C. Use for cryosectioning. Requires epitope/antigen retrieval.
- ethylene glycol and sucrose solution. Store in freezer between 4C and -20C. Use for cryosectioning. NO epitope/antigen retrieval. May get crystalization artifacts.
Size of Tissue for Cryosections
Embedding in OCT for Cryosectioning
Suggestion – Use flat rectangular glass (such as an oversized slide (do not used charged slide) or similar shape) with rounded/firepolished edges to protect tissue from damage.
- - use large glass baking dish filled with glycol cryoprotectant solution (will need to make) (need to find out what vendor uses so we can copy).
- Carefully empty the free-floating brain tissue into the glass dish and use a fine paint brush to gently float the tissue onto the glass plate.
- Lift the brain tissue and glass plate gently out of the liquid and take photographs. Determine the area of interest and remove the area of interest using scalpel.
- I propose the remaining tissue be placed back in cryoprotectant and frozen at -20 or -80 for long term storage
- The tissue has thawed at this point and should be embedded in OCT immediately. This can be kept in freezer until ready for cryosectioning.
- The tissue should not be allowed to dry at any point
- We still need to decide exactly how the location will be determined and confirm if we are only interested in CA1 of the hippocampus
- Will we be doing only 1 area of interest per sample?
- Will that area of interest fit in one OTC block and by a good size for cryosectioning? – needed for calculations as well as how and where to cut the native brain slice
OCT being added to a plastic embedding mold for crysectioning
Formalin-Fixed Paraffin-Embedded Technique
AbCam Paraffin-Embedded IHC
HOW TO DO IHC
