Four weeks after starting the treadmill running protocol in the treadmill and CIA + treadmill groups, all rats were sacrificed and right hind limbs were excised and fixed in 4% paraformaldehyde (Wako, Osaka, Japan), demineralized in 20% ethylenediaminetetraacetic acid, and embedded in paraffin. Sagittal sections, 6μm in thickness, were prepared from the center of the ankle. The sections were stained with hematoxylin and eosin or safranin O. We histologically evaluated arthritic changes, such as infiltration of inflammatory cells, synovial proliferation, destruction of articular cartilage, and bone erosion, as previously described [34 (link)]. To investigate the activity of osteoclasts and osteoblasts in vivo, sections were stained with a TRAP/ALP staining kit (Wako Pure Chemical Industries, Osaka, Japan) following the manufacturer’s procedure.
Trap alp staining kit
Manufactured by Fujifilm
Sourced in Japan
The TRAP/ALP staining kit is a laboratory product designed to identify and differentiate between two specific cellular enzymes: tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP). This kit provides the necessary reagents and protocols to perform cytochemical staining of these enzymes in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Tracp alp assay kit, by Takara Bio (2 mentions)
Formaldehyde, by Fujifilm (2 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Axiovert 200m, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Technovit 9100, by Kulzer (1 mentions)
Super cryoembedding medium, by Leica (1 mentions)
1 step nbt bcip substrate solution, by Thermo Fisher Scientific (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Methyl green solution, by Fujifilm (1 mentions)
Application suite v4, by Leica (1 mentions)
Multiskan sky, by Thermo Fisher Scientific (1 mentions)
Bz x800, by Keyence (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Nacalai Tesque (1 mentions)
Bz x700, by Keyence (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Bz 9000, by Keyence (1 mentions)
Rankl, by Fujifilm (1 mentions)
α mem, by Merck Group (1 mentions)
19 protocols using trap alp staining kit
1
Histological Analysis of Arthritic Changes
2
Maxillary Bone Analysis Protocol
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Rat maxillary bones were dissected and frozen in hexane cooled with carbon dioxide, followed by embedding in the media (Supercryo-embedding medium, SCEM; Leica Microsystems, Wetzlar, Germany). SCEM frozen blocks were covered with adhesive film (Cryofilm Type 2C; Leica) and coronally sectioned with the film at 5 μm intervals. Sections were then fixed in 10% neutral buffer formalin solution and stained with Mayer's hematoxylin and eosin. ALP activity was evaluated using a TRAP-ALP staining kit (Wako). To visualize bone double labeling, rat maxillary bones were dissected, fixed with 70% ethanol, and dehydrated in an ethanol series. Samples were then embedded with methyl methacrylate resin (Technovit9100; Heraeus-Kulzer, Hanau, Germany) and coronally sectioned every 40 μm (Kureha Special Laboratory, Tokyo, Japan). Sections were stained with toluidine blue and observed under an inverted optical microscope (Axiovert 200M; Carl Zeiss, Jena, Germany) using Axio Vision software (version 4.8; Carl Zeiss). The number of nuclei was counted on HE-stained images, at the suture, the osteogenic front, and the newly formed bone area (1000 × 1000-pixel box over the MPS).
3
Histological Assessment of Bone Remodeling
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The harvested block from another rabbit at 4 weeks after implantation was used. The paraffin-embedded tissues were decalcified, and cut into 4 µm sections, and stained with a TRAP/ALP staining KIT (Wako Pure Chemical Industries, Osaka, Japan) for histological assessment of osteoclasts and osteoblasts for new bone formation (Fig. 7 f).
4
Quantifying Bone Resorption and Formation
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Bone resorption and formation were assessed using a TRAP&ALP staining kit (Fujifilm Wako Pure Chemical Corp.) according to the manufacturer’s instructions. The number of multinucleated TRAP-positive cells and ALP-positive stained area (%) in the ROI were quantified and analyzed by a single examiner, and the average values were calculated.
5
Histological Analysis of Alveolar Bone
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For frozen sections, samples fixation was done in 4% PFA, decalcified with EDTA (10%), embedded in Tissue-Tek, and sectioned at 5 μm. Section staining was done using a TRAP/ALP staining kit (WAKO) following manufacturer instructions. We analyzed alveolar bone around the tensioned side of maxillary first molar mesiobuccal root was microscopically (Olympus Optical, Japan) analyzed. The unit area of positive staining in each section was calculated. Fixing of cultured cells was done in PFA (4%) for 10 min 7 days after osteogenic induction and stained with 1-Step™ NBT/BCIP substrate solution (Thermo Scientific). Images were taken randomly, and ALP positive area of each group calculated on ImageJ.
6
Decalcification and Sectioning of Molar Tissues
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Upper 1st molars with surrounding tissue were dissected, decalcified with 10% (w/v) EDTA for 3 weeks at 4°C, and embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan) or paraffin according to a standard protocol (Kaku et al., 2016 (link); Mizukoshi et al., 2021 (link)). Sagittal cryosections (10-µm thick) were prepared on membrane-coated slides (Leica Biosystems, Wetzlar, Germany), and 5-µm thick sagittal paraffin sections were prepared using a microtome (REM-710+MC-802C; Yamato Kohki, Saitama, Japan). Paraffin sections were stained with hematoxylin and eosin and a tartrate-resistant acid phosphatase/alkaline phosphatase (TRAP/ALP)-staining kit (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) according to the manufacturer’s instructions.
7
Histochemical Analysis of GTCB-OCs
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FFPE sections (not exposed to denosumab) were deparaffinized, rehydrated, and incubated with adjusted solutions for 15 min at 37℃ according to the manufacturer's protocol (TRAP/ALP staining kit, Fujifilm Wako) for the detection of TRAP activity of GTCB-OCs. The stained sections were washed three times with deionized water and counterstained with Methyl Green Solution (FUJIFILM Wako) for 5 s. Denosumab-exposed tissue samples were also stained as negative controls.
8
Histochemical Analysis of Bone and Tissue
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Paraffin-embedded sections of decalcified femurs were subjected to tartrate-resistant
acid phosphatase (TRAP) staining by using a TRAP/ALP staining kit (Wako Pure Chemical
Industries, Osaka, Japan). The percentage of bone surface covered with osteoclasts was
calculated from TRAP-stained sections by using the Leica Application Suite V4.5 software
(Leica, Wetzlar, Hesse, Germany). Histochemical analyses for COX and succinate
dehydrogenase activities were performed as described previously [18 (link)] by using cryosections (thickness, 10 µm) of renal
or duodenal tissue. Haematoxylin and eosin–stained sections were used for
histopathological analysis of renal tissue.
acid phosphatase (TRAP) staining by using a TRAP/ALP staining kit (Wako Pure Chemical
Industries, Osaka, Japan). The percentage of bone surface covered with osteoclasts was
calculated from TRAP-stained sections by using the Leica Application Suite V4.5 software
(Leica, Wetzlar, Hesse, Germany). Histochemical analyses for COX and succinate
dehydrogenase activities were performed as described previously [18 (link)] by using cryosections (thickness, 10 µm) of renal
or duodenal tissue. Haematoxylin and eosin–stained sections were used for
histopathological analysis of renal tissue.
9
Osteoclast Differentiation Quantification
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RAW264.7 or Opto-RANK cells were differentiated as described above. Cells in 96-well plates were fixed and stained with the TRAP/ALP Staining Kit (FUJIFILM Wako Pure Chemicals), according to the manufacturer’s instructions. For experiments shown in Fig. 4 , cells were stained with 4',6-diamidino-2-phenylindole (0.5 μg/mL, Nacalai Tesque). Brightfield and fluorescence images of the cells were captured using BZ-X700 and BZ-X800 microscopes (Keyence, Osaka, Japan). TRAP activity in the cell lysate was assessed using the TRACP & ALP Assay Kit (TaKaRa Bio), according to the manufacturer’s instructions. Absorbance was measured at 405 nm using a Multiskan Sky microplate spectrophotometer (Thermo Fisher Scientific).
10
TRAP Staining of Tissue Sections
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TRAP staining of the sections was carried out using the TRAP/ALP staining kit (Fujifilm WAKO, Japan) according to the manufacturer’s instructions. The number of TRAP-positive cells was estimated and expressed as cell numbers per millimeter in the MPS (Image-pro Plus, Media Cybernetics, USA).
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