Non-decalcified sample protocol (for simple staining)

Sample fixation and dehydration

1. Fix the sample in 10% formalin at room temperature for 3 days.

2. Remove the sample from formalin, cut the sample as small as possible, and put it in a tea
bag.
3. Put the teabag into serial alcohol.
a. 50% alcohol 6 hours
b. 70% alcohol 6 hours
c. 80% alcohol 6 hours
d. 90% alcohol 6 hours
e. 100% alcohol (1) 1 day
f. 100% alcohol (2) 1 day
4. Then, Transfer the maxilla to xylene and leave it overnight for thorough clearing.

Methyl methacrylate preparation

1. Mix methyl methacrylate (MMA) with Sodium hydroxide (NaOH) and Sodium chloride
(NaCl). This step is to remove the inhibitor from the MMA. No specific amount of each
solution.
2. Wait until the solution separates, taking only the upper part of the solution (MMA)
3. Repeat step 1 for 2-3 times or until the solution is clear.

Infiltration and embedding

1. Remove the sample from the tea bag.

2. Mix the MMA with dibutyl (n-butyl) phthalate (8.5 : 1.5)
3. Put the sample into the solution for 2 days in 4o Celsius refrigerator.
4. Mix the MMA with dibutyl (n-butyl) phthalate (8.5 : 1.5), then add 0.1g benzoyl peroxide
per 10 mL of solution using a plastic spoon.
5. Put the sample into the solution for 2 days in 4o Celsius refrigerator.
6. Mix the MMA with dibutyl (n-butyl) phthalate (8.5 : 1.5), then add 0.25g benzoyl
peroxide per 10 mL of solution using a plastic spoon.
7. Mix the MMA with dibutyl (n-butyl) phthalate (15.3 : 2.7), then add 0.45g benzoyl
peroxide per 18 mL of solution using a plastic spoon. Finally, add
N, N-Dimethylaniline 60 μl per 18mL of solution.
8. Embed the sample into the solution in a mold block at room temperature.
9. Wait 2 days or until the solution becomes hard. (Resin will turn red-brown)

Note: Function of:

- dibutyl (n-butyl) phthalate: serves as a softening agent, making the methyl methacrylate mixture more pliable and easier to
handle. It helps to reduce brittleness and increase the flexibility of the embedding resin, allowing easier sectioning of the
embedded tissue. The presence of dibutyl phthalate helps prevent excessive hardness and brittleness in the embedding resin,
making it easier to trim excess resin and obtain a suitable block shape for sectioning.
- benzoyl peroxide: initiates the polymerization reaction of the methyl methacrylate monomer when it comes into contact with
the accelerator and is exposed to suitable conditions (e.g., heat).
- N, N-Dimethylaniline: enhances the polymerization reaction initiated by benzoyl peroxide.
 Non-decalcified sample protocol (for immunohistochemistry) Technovit 9100

Sample fixation and dehydration

1. Fix the sample in 10% formalin at room temperature for 3 days.

2. Remove the sample from formalin, cut the sample as small as possible, and put it in a tea
bag.
3. Put the teabag into serial alcohol.
a. 50% alcohol 6 hours
b. 70% alcohol 6 hours
c. 80% alcohol 6 hours
d. 90% alcohol 6 hours
e. 100% alcohol (1) 1 day
f. 100% alcohol (2) 1 day
4. Then, Transfer the maxilla to xylene and leave it overnight for thorough clearing.

Infiltration and embedding

 Sample mounting and cutting

1.