TRAP staining kit

2-8 ℃, avoid light

1. The validity period is the period during which the reagent kit is stored under the required conditions before being opened.
2. Please use up the reagent as soon as possible after unpacking!

3 months (note: the incubation solution inside is prepared in one go and will be effective within a week!)

microscope

1. Paraffin section
2. Frozen section
3. Cell smear, slide film
4. Blood smear, etc

1 Optical microscope
2. Dyeing vat
3. Pipette
4. Stopwatch
5. Ice box

pure water

1. Glass slide
2. Suction head
3. Disposable gloves

1. TRAP incubation solution is prone to failure, and this method should use skin puncture blood smear. After drying, it should be stained in a timely manner;
When staining frozen sections, the exposure time of the sections at room temperature should be reduced.
3. The sample should be fresh and processed immediately after collection, otherwise it will affect the activity of the enzyme.
4. The organization fixation should be carried out in a refrigerator at 4 ℃ for no more than 24 hours, otherwise the enzyme activity will weaken or disappear.
5. It is generally not recommended to slice with paraffin. If paraffin sectioning is required, the temperature of the tissue during paraffin embedding should not exceed 56 ℃. Paraffin wax with a melting point of 52-54 ℃ should be used for wax immersion, and the immersion time should be short, otherwise the enzyme activity will weaken or disappear.
6. Paraffin sections do not require fixation.

(Note: The incubation solution inside is prepared in one go and will be effective within one week!)
Attention: For other working fluids, please prepare them fresh before use and use them immediately after preparation. Do not store them after one use.

Staining treatment of samples:
1、 Blood smear and bone marrow smear
1. Push the video:
Take about 3ul of whole blood or bone marrow and place it on a glass slide. Hold the slide at a 30 ° angle to the slide and place it directly in front of the blood droplet. Move it back slightly to come into contact with the blood droplet. The blood droplet will spread along the lower edge of the slide, and then slide steadily forward along the plane of the slide at a constant speed until the blood droplet has covered the blood film. Don't be too thin to avoid too few cells, and don't be too thick to avoid too much overlap.
2. Fixed:
Allow the smear to air dry naturally, then place it in fixative A in this kit and fix for 2-3 minutes. Rinse with water for 30-60 seconds.
3. Incubation:
After washing with water and before drying, place it in the substrate incubation solution and incubate in the dark for 60 minutes. The dyeing effect will deteriorate if it is too dry.
4. Re dyeing:
After incubation, remove and wash with water for 1 minute, and perform counterstaining on the slide before it dries. You can counterstain with hematoxylin for 1-2 minutes, or counterstain with methyl green for 2-3 minutes, either of which is acceptable.

2、 Cell smear and cell slide
1. Fixed:
Place the cell smear or cell slide into fixative A in this kit and fix for 2-3 minutes, then wash with water for 30-60 seconds.
2. Incubation:
After washing with water and before drying, place it in the substrate incubation solution and incubate in the dark for 60 minutes. The dyeing effect will deteriorate if it is too dry.
3. Re dyeing:
After incubation, remove and wash with water for 1 minute, and perform counterstaining on the slide before it dries. You can counterstain with hematoxylin for 1-2 minutes, or counterstain with methyl green for 2-3 minutes, either of which is acceptable.

3、 Frozen section
1. Return temperature:
Place the frozen slices stored in the -20 ℃ freezer on the slicing rack and let them warm for 5-10 minutes. It can be placed in a 37 ℃ incubator for reheating, or the slicing rack can be placed in a small box and the box can be placed in a 37 ℃ water bath for reheating.
2. Hydration:
Soak the warmed slices in water for 1-2 minutes.
3. Fixed:
Let the slices dry naturally in the air, then place them in the fixative in this kit and fix for 2-3 minutes. Rinse with water for 30-60 seconds.
4. Incubation:
After washing with water and before drying, place it in the substrate incubation solution and incubate in the dark for 60 minutes. The dyeing effect will deteriorate if it is too dry.
5. Re dyeing:
After incubation, remove and wash with water for 1 minute, and perform counterstaining on the slide before it dries. You can counterstain with hematoxylin for 1-2 minutes, or counterstain with methyl green for 2-3 minutes, either of which is acceptable.

4、 Paraffin section
1. Dewaxing:
Remove wax in xylene for 5-10 minutes. Replace with fresh xylene dewaxing for 5-10 minutes.
2. Soaking:
Soak in anhydrous ethanol for 5 minutes. Then use 90% and 70% ethanol for 2 minutes each.
3. Hydration:
Soak in distilled water for 2 minutes.
4. Incubation:
Incubate in the substrate incubation solution in the dark for 60 minutes before it dries. The dyeing effect will deteriorate if it is too dry.
5. Re dyeing:
After incubation, remove and wash with water for 1 minute, and perform counterstaining on the slide before it dries. You can counterstain with hematoxylin for 1-2 minutes, or counterstain with methyl green for 2-3 minutes, either of which is acceptable.

Result analysis:
Positive reactions are bright red or dark red particles located in the cytoplasm.

Is cell induction unsuccessful?
The quality of cytokines is crucial when inducing osteoclasts in cell lines or primary BMM cells. When the induction effect is not good, it can be considered to adjust the induction conditions, increase the concentration of RANKL, or try adding M-CSF.

How to determine positive cells?
Generally, RAW cells are very diverse in morphology, and morphological changes do not necessarily indicate a problem. It is generally believed that multinucleated osteoclasts can only be considered multinucleated when they are TRAP positive.

1.Guiping Chen et al.
Bergapten suppresses RANKL-induced osteoclastogenesis and ovariectomy-induced osteoporosis via suppression of NF-κB and JNK signaling pathways
Biochemical and Biophysical Research Communications 2019

2.Wen‑Yong Fei et al.
Magnolol prevents ovariectomy‑induced bone loss by suppressing osteoclastogenesis via inhibition of the nuclear factor‑κB and mitogen‑activated protein kinase pathways
International Journal of Molecular Medicine 2019