Double staining of bone-related enzymes (TRAP, ALP) for paraffin slices.

1.It is an
to study the physiological activity of related cells to understand the metabolic state of living bones. Bone formation of bone cells can be understood after alkaline
phosphatase
(ALP) staining of bone
tissue
, and bone tissue anti-salt acid phosphatase (TRAP) staining can be used to understand bone absorption of bone-breaking cells. Double-staining
the
the same slice can provide an understanding of both. In addition, it has the advantage of being in the decalcification specimen, as long as it can be double-stained, without the need for special equipment, only with general equipment can easily observe bone metabolism. Under this premise, we need to study the feasibility of double staining.
2.Production of standard specimens
the use of resin encumbase or frozen slices of legally made specimens as standard specimens, compared with calcium decalcification paraffin slices. That is, after the alcohol is fixed to the elbows of mice, it is encumerated with glycol methyl acrylates (GMA), sliced into 2 m using a slicing mechanism used in hard tissue, and then stained with ALP. Then, make a calcium-frozen slice. After that, the feasibility of double staining in various stages of fixation, decalcturing and dyeing was studied and improved.
photo is a color-stained specimen
the
of the mouse as a standard specimen of this experiment. As a positive control, the dyeing and dyeing were evaluated together with the decalcite wax slice.
soaked in the lumbar spine and upper right limbs of mice decalculfurated citric acid with 30% of the sugar, then buried by the OCT complex, frozen by Tissue-Tek PINO (Sakura Finetek Japan Co., Ltd. products), and cut by Sakura Finetek Japan Co., Ltd. products, resulting in a 5 m frozen slice. The slices are dyed with enzymes. Not only can TRAP dyeing and ALP dyeing monochrome, but also double staining.
staining study
(1) was not fixed by Formarin, only alcohol for a second fixed sample, ALP staining showed strong positive. TRAP staining is negative. (2) trap and ALP can be dyed well after 16 hours of fixation by Formarin and (3) is fixed for 4 days. However, the clinical sample (bone cartilage swelling) after one month's fixation by Formarin showed positive for TRAP staining, but ALP could not be dyed.
decalcification solution decalcification 3 days (Histra-DC, 8 to 16 degrees C) after
rats
knee joint. Neither TRAP nor ALP can be dyed. In contrast, the middle citrate decalcification solution and the far right
EDTA
decalculum solution can decalcide the sample after TRAP/ALP double staining.
3.About fixed
ALP staining uses fresh materials that are not fixed by Formarin, so if they are not fixed for a short period of time, their enzyme activity is lost. We recommend using 80% alcohol to fix it. Please verify that
(1) the knee joint of the mouse is not fixed for the first time, and the second fixation group is directly soaked with alcohol.
(2) after the first 16 hours of fixation of the mice's lumbar spine by Formarin (4% polyformaldehyde solution), the second fixed group was carried out with alcohol.
(3) after the first fixed mouse lumbar vertebrae by Formarin for 4 days, and then the second fixed group.
(4) clinical samples were first fixed in the mice's lumbar vertebrae by Formarin for one month before a second fixed group was performed.
the double staining of TRAP and ALP in each group, and to test the feasibility of dyeing. In this experiment, Formarin was able to double-stain TRAP and ALP for a fixed time of 16 hours to 4 days.
4.About decalcification
ALP is a protein containing metals (Zn). Decalcification causes Zn to also be removed, causing the enzyme to become inactive. To compensate for this shortcoming, zinc sulfate (ZnSO4) is added to make the ALP active. Add 0.4 ml of 1% ZnSO4 solution to each 100 ml calcium solution to replenish Zn. In addition, while adding Zn to the citrate buffer as a decalculfur solution, the relevant research on adding the same amount of chelating agent EDTA solution is in the process. There have also been studies on whether enzymes in acidic decalculfurum solutions (foretic acid formarin solutions) can react. The results showed that the calcium decalculfuration solution, like citric acid, was well dyed in the EDTA solution. Conversely, enzymes cannot be dyed in the foreric acid formarin solution. In addition, the decalcification method is to use ultrasonic decalcturing device (Histra-DC, normal lunaturum) at a low temperature of 8-16 degrees C, continuous operation for 3-6 days, so that the sample decalcification. After calcium decalcification, wash with glycine and Veronal buffer (pH7.4) and adsorption on the tissue with calcium phosphate to prevent precipitation.
5. About staining
is using Loch's Gomori method. This experiment used a TRAP/ALP staining
-reagent
box (and photo-pure medicine, product number 294-67001) using both acetox staining and a-coupled reaction. With regard to the thickness of the slices, Lorch recommends 8 m, and has also studied whether ordinary 4 m slices can be dyed, double-stained order should first dye which of TRAP and ALP, sealing agents must choose water-soluble sealing tablets and so on.
the result of the dyeing method is that in the amerinus dyeing method, the slice is too thick will have the phenomenon of enzyme diffusion, that is, the bone substation TRAP dyeing tendency to dye red. Even slices of 4 m can be fully dyed as long as the reaction conditions (reaction temperature and reaction time) are enhanced. In other words, trap dyeing reaction temperature is room temperature to 37 degrees C, reaction time is 30 minutes to 45 or 60 minutes. ALP staining can be performed for 45 minutes to 3 hours at 37 degrees C or extended at room temperature (10 to 15 degrees C) to one night. Finally, we can get the two tone balance, good dyeing results.
, enhanced reaction conditions cause more pigment particles to be produced on ALP-stained slices. The dyeing order between TRAP and ALP can start on either side. However, when ALP and then TRAP dyeing, the positive site appears significantly red, using fresh bone-breaking cell staining, can get relatively good specimens. But the ALP's reaction product produces white turbid particles from the TRAP solution, which precipitate in tissues. Therefore, first TRAP dyeing and then ALP dyeing words will be better. Seal: with methyl green, a few seconds can make the nuclear dyeing, washing at 37 degrees C
dying
, transparent by xylene, by the permanent seal of cannabinol (Marinol). Some literature suggests dehydration with alcohol before transparency, but this can leach and spread reaction products.
used EDTA decalcification paraffin slices from the lumbar spine of 1-year-old mice for TRAP/ALP double staining. The TRAP staining of bone-breaking cells is red, and the ALP of cells with strong cell activity (osteoblasts, cartilage cells) is brown. (Above: Weak expansion, below: Strong expansion)
6. Conclusion
TRAP and ALP two enzymes in each fixed, decalcturing, dyeing, as long as after a number of improvements, can make decalcturing paraffin specimen dyeing. However, enzyme diffusion is more pronounced, especially when TRAP is dyed. There are many factors, mainly should be affected by fixed, if not fixed enough, calcium decalcturing is prone to problems, resulting in enzyme diffusion phenomenon. In addition, attention must be paid to the effects of decalcification itself, that is, compared with non-calcium decalcification specimens, decalcification specimens observed more diffusion phenomenon. It is indicated that decalcification of specimens is likely to lead to the spread of enzymes. In addition, the thickness of the slices or the order of double staining should also be considered. We will study in this direction in the future.
article was
Yuanyuan, Second Research Office of Plastic Surgery Spine Surgery, University of Tokyo Medical Department affiliated
.