Paraffin Embedding Protocol
The aim of this protocol is to provide instructions for paraffin embedding of fixed, cell laden constructs. Embedded samples can, among other applications, be stained for immunofluorescence and immunohistology analysis. Follow Fixation Protocol before starting this protocol.
- Dry oven at 58°C
- Fixed, cell laden construct
- Embedding cassettes
- 70% Ethanol
- 96% Ethanol
- 100% Ethanol
- Xylene or xylene substitute, e.g. Shandon Xylene Substitute (Thermofisher, Ref: 9990505)
- Tissue embedding machine
1. Preparation of paraffin
Fill ¾ of a suitable container with paraffin and put in the 58°C dry oven to melt.
Note: This may take several hours to melt. Do not increase the temperature of the oven, higher temperatures will make the paraffin hard and brittle.
2. Preparation of constructs
Put fixed, cell laden constructs in embedding cassettes and label the cassette properly with a pencil.
Follow following dehydration series either through 1) moving the cassettes with constructs between beakers with the different reagents or 2) by adding and removing the different reagents of the dehydration series to a beaker with the cassettes.
Handle both xylene and ethanol with care inside a fume hood with proper PPE.
- 70% ethanol: 2 x 30 min
- 96% ethanol: 2 x 30 min
- 100% ethanol: 2 x 30 min
- Xylene or xylene substitute: 3 x 30 min
Note 1: If fixed samples have been stored in 70% ethanol before embedding only 1 x 30 min of 70% is necessary.
Note 2: The constructs might shrink after the dehydration procedure depending on the bioink type.
4. Paraffin infiltration
Transfer cassettes with constructs to the beaker with melted paraffin. Let sit in the oven for 45 min.
Note 1: The transfer of the cassettes to the melted paraffin must be done quickly since the paraffin solidifies under 56°C.
5. Paraffin embedding
Add the infiltrated samples to the cassette container of the tissue embedding machine.
If samples have been stored in room temperature before embedding let the excess paraffin melt away from the cassettes by leaving them in the cassette holder pocket for ~20 min. If proceeding directly from infiltration, step 4, you can start embedding after a few minutes since the paraffin is already warm.
To embed samples:
- Open the cassette.
- Fill a metallic embedding mould with paraffin.
- With warm tweezers; transfer the constructs. from the cassette to the mould with paraffin and push the constructs (cross-section down) to the bottom of the mould.
- Let stiffen slightly on a cold plate so the sample stays at the bottom of the mould.
- Add the lid of the embedding cassette on the top of the mould before the paraffin stiffens completely. Throw the bottom.
- Leave the embedded construct at the cooling plate until it easily can be removed from the mould (~20 min).
Note 1: Don’t leave the mould with the embedded construct at the cooling plate for too long, if the paraffin gets too cold it can break.
Note 2: The paraffin embedded blocks can be stored at room temperature for an extended period of time.