Method of fixating tissue sample using propolis-containing composition

2:

• The method of claim 1, wherein the propolis extract is present in the propolis-containing composition at a concentration of 1 to 15 wt. %, based on the total weight of the propolis-containing composition.

3:

• The method of claim 1, wherein the propolis extract further comprises at least one substance selected from the group consisting of an oil, a pollen, an amino acid, a minerals, a sugar, are B vitamin, a C vitamin, an E vitamin, a flavonoid, a phenol, an aromatic compound, and combinations thereof.

4:

• The method of claim 3, wherein the propolis extract further comprises at least one substance selected from the group consisting of luteolin, 3-O—[(S)-2-methylbutyroyl]pinobanksin, 6-cinnamylchrysin, 8-[(E)-4-phenylprop-2-en-1-one]-(2R,3S)-2-(3,5-dihydroxyphenyl)-3,4-dihydro-2H-2-benzopyran-5-methoxyl-3,7-diol, 8-[(E)-4-phenylprop-2-en-1-one]-(2S,3R)-2-(3,5-dihydroxyphenyl)-3,4-dihydro-2H-2-benzopyran-5-methoxyl-3, 7-diol, and 8-[(E)-4-phenylprop-2-en-1-one]-(2R,3S)-2-(3-methoxyl-4-hydroxyphenyl)-3,4-dihydro-2H-2-benzopyran-5-methoxyl-3,7-diol.

7:

• The method of claim 1, wherein the at least one liquid comprises one or more solvent selected from the group consisting of water, alcohol, glycol, glycerol, glycerin, glyceryl ether, diglycerol, and vegetable oil.

8:

• The method of claim 1, wherein the at least one liquid comprises propylene glycol and vegetable glycerol, and wherein a volume ratio of the propylene glycol to the vegetable glycerol is in a range of 20:1 to 1:20.

9:

• The method of claim 1, wherein the propolis-containing composition is in the form of at least one selected from the group consisting of a solution, an oil-in-water emulsion, a micro-emulsion, a capsule suspension, and a dispersion.

10:

• The method of claim 1, wherein the at least one liquid comprises propylene glycol and water, and wherein the propolis-containing composition is an oil-in-water emulsion having an average particle size in a range of 5 to 200 nanometers (nm).

11:

• The method of claim 1, wherein the propolis-containing composition further comprises one or more additives selected from the group consisting of a fixative agent, an emulsifying agent, a buffering agent, a penetration enhancer, a crosslinking agent, a decalcifying agent, a salt, and a stabilizer.

12:

• The method of claim 11, wherein the fixative agent is at least one selected from the group consisting of formalin, glutaraldehyde, honey, syrup, and jaggery.

13:

• The method of claim 11, wherein the emulsifying agent is at least one selected from the group consisting of a cationic surfactant, an anionic surfactant, a non-ionic surfactant, and an amphoteric surfactant.

14:

• The method of claim 11, wherein the emulsifying agent comprises a fatty alcohol sulfate, a fatty alcohol polyoxyalkylene ether, and a fatty acid alkanolamide.

15:

• The method of claim 11, wherein the buffering agent is at least one selected from the group consisting of sodium phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

16:

• The method of claim 11, wherein the salt comprises at least one alkali metal halide selected from the group consisting of sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide, and potassium iodide.

17:

• The method of claim 11, wherein the decalcifying agent comprises ethylenediaminetetraacetic acid (EDTA).

18:

• The method of claim 1, wherein the treating is for 1-72 hours, and forms a fixated tissue having enhanced preservation and fixation ability compared to a tissue sample treated with formalin.

The method of claim 1, wherein a ratio of the volume of the tissue sample to the volume of the propolis-containing composition is in a range of 1:1 to 1:20.

20:

• The method of claim 1, further comprising:
• staining the fixated tissue sample with at least one stain selected from the group consisting of a hematoxylin and eosin stain, a reticulin stain, a trichrome stain, a periodic acid-schiff stain, a desmin stain, a TTF1 stain, a CD3 stain, and a PAX8 stain.