Paraffin Embedding Preparation
56.6g - Paraffin
3.5g - Stearic Acid(SA)
0.35g - Sudan IV dye
20g - Vybar (for stiffening)
***melt at 75ºC
  1. Mix wax and vybar together in a single beaker and incubate in oven at 75ºC for at least four hours.
  2. 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.
  3. 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.)
  4. Wait a few hours before use to ensure that all of the remaining particles have settled at the bottom.
  5. 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)

  1. Remove formalin fix from scintillation vials or microvials containing embryo/heart sample into waste beaker.
  2. 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.
  3. Proceed to 15 minute wax exchanges.
  4. 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
  1. Turn on cooling plate of embedding station till it turns on.
  2. Wait till temperature decreases till -2ºC.
  3. Heat metal molds on paraffin plate.
  4. Take out melted wax from oven.
  5. Pour melted wax in metal mold till half full.
  6. Take out vial with sample from oven.
  7. Remove sample with spatula and place sagitally in the metal mold, making sure the sample lies in the center of the metal mold.
  8. Place labeled green cassette on top.
  9. Pour more wax on top of the cassette.
  10. Wait 30 seconds, and move to the cooling element of embedding station.
  11. Place in cold room overnight for it to harden.