Paraffin Embedding Preparation
56.6g - Paraffin
3.5g - Stearic Acid(SA)
0.35g - Sudan IV dye
20g - Vybar (for stiffening)
***melt at 75ºC
- Mix wax and vybar together in a single beaker and incubate in oven at 75ºC for at
least four hours.
- In a small vile place SA and dye (shake vial for a more evenly melted mixture);
then incubate in oven for at least 4 hours.
- Once everything is melted pour the SA/dye mixture into wax and stir. (The dye will
not completely dissolve in the wax and you don’t want to embed using this part of
the wax.)
- Wait a few hours before use to ensure that all of the remaining particles have settled
at the bottom.
- Reheat wax at 75ºC when it’s time to embed.
Dehydration Processing of Embryo Samples
(Note: Timing will vary based on age of sample and sample type)
- Remove formalin fix from scintillation vials or microvials containing embryo/heart
sample into waste beaker.
- Use spatula to remove heart or full embryo and place in scintillation vial containing
the designated solution for the desired processing time. (Refer to tables below.
- Proceed to 15 minute wax exchanges.
- Incubate in wax overnight (O/n) at 75º C.
Table 1: Age-Specific Durations for Dehydration Processing of Mouse Embryo Samples
|
|
9.5-10.5d
|
11.5-12.5-13.5d
|
14.5-15.5d
|
16.5d
|
17.5 - 18.5d
(Skin before
processing)
|
Newborn
(Skin before
processing)
|
|
70%
|
45 min
|
30 min
|
1-2 hours
|
1 hr 10 min
|
1-2 hrs
|
1-2 hrs
|
|
95%
|
30 min
|
30 min
|
35 min
|
35 min
|
1 hr
|
1 hr 20 min
|
|
95%
|
30 min
|
30 min
|
35 min
|
35 min
|
1 hr
|
1 hr 20 min
|
|
100%
|
30 min
|
30 min
|
35 min
|
35 min
|
1 hr
|
1 hr 20 min
|
|
100%
|
30 min
|
20 min
|
35 min
|
35 min
|
1 hr
|
1 hr 20 min
|
|
100%
|
30 min
|
20 min
|
35 min
|
35 min
|
1 hr
|
1 hr 20 min
|
|
Xylene
|
20 min
|
20 min
|
35 min
|
35 min
|
1 hr
|
1 hr
|
|
Xylene
|
20 min
|
15 min
|
35 min
|
35 min
|
1 hr
|
1 hr
|
|
Wax
|
15 min
|
15 min
|
15 min
|
15 min
|
15 min
|
15 min
|
|
Wax
|
15 min
|
15 min
|
15 min
|
15 min
|
15 min
|
15 min
|
|
Wax
|
4 hours
|
4 hours
|
O/n
|
O/n
|
O/n
|
O/n
|
Table 2. Age-Specific Durations for Dehydration Processing of Heart or Heart/Lung
Samples from Mouse Embryos
|
|
13.5d, 15.5d, 16.5d
|
17.5d, 18.5d, neonates
|
|
70%
|
40 min
|
1 hr
|
|
95%
|
40 min
|
1 hr
|
|
95%
|
40 min
|
1 hr
|
|
100%
|
40 min
|
1 hr
|
|
100%
|
40 min
|
1 hr
|
|
100%
|
40 min
|
1 hr
|
|
Xylene
|
15 min
|
1 hr 20 min
|
|
Xylene
|
15 min
|
1 hr 20 min
|
|
Wax
|
15 min
|
15 min
|
|
Wax
|
15 min
|
15 min
|
|
Wax
|
O/n
|
O/n
|
Embedding Sample
- Turn on cooling plate of embedding station till it turns on.
- Wait till temperature decreases till -2ºC.
- Heat metal molds on paraffin plate.
- Take out melted wax from oven.
- Pour melted wax in metal mold till half full.
- Take out vial with sample from oven.
- Remove sample with spatula and place sagitally in the metal mold, making sure the
sample lies in the center of the metal mold.
- Place labeled green cassette on top.
- Pour more wax on top of the cassette.
- Wait 30 seconds, and move to the cooling element of embedding station.
- Place in cold room overnight for it to harden.