TRAP staining was performed in whole-mount 3-month-old fish (3 mpf) (control, n=4; kita-RAS, n=5) using an Acid Phosphatase, Leukocyte (TRAP) Kit (Merck, 387A), following the instructions provided by the manufacturer. Fish were fixed overnight in fixative solution (provided). Samples were washed for 15 min in distilled water, followed by permeabilisation using 1% trypsin in 30% borate solution at 37°C overnight. Fish were incubated in TRAP staining solution (provided) at 37°C for 6 h in the dark, followed by two washes of 10 min each in distilled water. Pigmentation was removed by incubating the specimens in 3% H2O2. Pictures were taken from dissected spines placed in 70% glycerol under a Leica stereomicroscope. Quantification of TRAP signal was performed using Fiji (Schindelin et al., 2012 (link)). Images were converted to 32-bit and LUT (physics) applied. We inverted the LUT, flattened the images and calculated the mean of red pixels, corresponding to high TRAP signal.
Trap kit
Manufactured by Merck Group
Sourced in United States, Germany
The TRAP kit is a laboratory equipment product manufactured by Merck Group. It is designed to perform Tartrate-Resistant Acid Phosphatase (TRAP) assays, which are used to measure the activity of this enzyme in biological samples.
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Lab products found in correlation
M csf, by R&D Systems (8 mentions)
Rankl, by Thermo Fisher Scientific (7 mentions)
Rankl, by R&D Systems (7 mentions)
M csf, by Thermo Fisher Scientific (4 mentions)
α mem, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Stereomicroscope, by Leica camera (2 mentions)
Osteoassay, by Corning (2 mentions)
Alizarin red solution, by Merck Group (2 mentions)
Ix51 microscope, by Olympus (2 mentions)
F ab 2 goat anti mouse igg h l af647, by Thermo Fisher Scientific (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
M csf, by Merck Group (2 mentions)
Rankl, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dm4000b, by Leica camera (1 mentions)
Diaminobenzidine dab, by Vector Laboratories (1 mentions)
Anti osterix, by Abcam (1 mentions)
Anti osteocalcin, by Takara Bio (1 mentions)
138 protocols using trap kit
1
TRAP Staining in Zebrafish Spine
2
Quantifying Inflammation-Driven Osteoclastic Activity
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To quantify the histological sections, samples were evaluated by double-blinded examiners as described earlier83 (link). Inflammatory cell infiltration (ICI) was scored on a scale of 0 through 5, with 0 corresponding to no sign of inflammation, 1 corresponding to incipient ICI in the derma/subderma, 2 corresponding to mild ICI in the derma/subderma, 3 corresponding to moderate ICI in the derma/subderma and scarce inflammation in the surrounding bone, 4 corresponding to advanced ICI in the derma/subderma and mild inflammation in the surrounding bone, and 5 corresponding to severe ICI in the derma/subderma and advanced inflammation in the surrounding bone.
To analyze the inflammation-mediated osteoclastic activity, a tartrate-resistance acid phosphatase (TRAP) assay was performed with TRAP Kit (387A-1KT, Merck, France). The TRAP solution was prepared according to manufacturer’s instructions. The histological slides were immersed into freshly prepared TRAP solution and kept for 1 h at 37 °C avoiding any exposure to light. Subsequently, the slides were washed with deioinized water and rinsed with tap water before observing under the microscope (Leica DM4000B, France).
To analyze the inflammation-mediated osteoclastic activity, a tartrate-resistance acid phosphatase (TRAP) assay was performed with TRAP Kit (387A-1KT, Merck, France). The TRAP solution was prepared according to manufacturer’s instructions. The histological slides were immersed into freshly prepared TRAP solution and kept for 1 h at 37 °C avoiding any exposure to light. Subsequently, the slides were washed with deioinized water and rinsed with tap water before observing under the microscope (Leica DM4000B, France).
3
TRAP Staining of Osteoclasts in Zebrafish
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TRAP staining was performed in whole-mount 3 month old fish (3mpf) (control, n = 4; Kita-RAS, n = 5) using Acid Phosphatase, Leukocyte (TRAP) kit (Merck, cat 387A) and following the instructions provided by the manufacture. Fish were fixed overnight in fixative solution (provided). Samples were washed for 15 min in distilled water, followed by permeabilization using 1% trypsin in 30% borate solution at 37˚C overnight. Fish were incubated in TRAP staining solution (provided) at 37˚C for 6h in the dark, followed by two washes of 10 min each in distilled water. Pigmentation was removed by incubating the specimens in 3% H 2 O 2. Pictures were taken from dissected spines placed in 70% glycerol under a Leica stereomicroscope. Quantification of TRAP signal was performed using Fiji (Schindelin et al., 2012) . Images were converted to 32-bit, and LUT (physics) applied. We inverted the LUT, flattened the images and calculated the mean of red pixels, correspondent to high TRAP signal.
4
Osteoclastogenesis and Bone Resorption Assay
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At the end of the osteoclasts differentiation protocol (day 12), culture medium was removed and cells were fixed with 4% formaldehyde for 5 minute and stained with leukocyte acid phosphatase (TRAP) kit (Sigma-Aldrich) according to the manufacturer's instructions. Stained positive cells (>3 nuclei) were then counted. Osteoclast activity was assessed by culturing cells on plates coated with a synthetic inorganic bone mimetic matrix (Osteoassay, Corning). At day 12 the culture medium was removed and plates filled with sodium hypochlorite solution to evaluate the ability of mature osteoclasts to reabsorb this substrate; the pits produced by osteoclasts were quantified by ImageJ software [27 (link)].
5
Quantifying Osteoblast and Osteoclast Markers in Diabetes
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Tissue sections (n=20 in non-diabetic and diabetes groups, respectively; specimens were randomly selected; n=20 in control group) from lateral tibial plateau were evaluated using immunohistochemistry as described previously.14 (link),17 (link) In brief, biomarker of the osteoclasts (tartrate-resistant acidic phosphatase, TRAP) was detected using TRAP staining with a commercial TRAP kit (Sigma-Aldrich, Missouri, USA). To detect biomarkers of osteoblasts (osteocalcin) and osteoprogenitors (osterix),14 (link),17 (link) sections underwent heat-induced antigen retrieval in citrate buffer, followed by incubation with either anti-osterix (Abcam, Cambridge, UK) or anti-osteocalcin (TakaRa, Shiga, Japan) primary antibodies overnight. Next, horseradish peroxidase-labeled secondary antibodies (Abcam) was added and incubated for 60 min. Color was developed using diaminobenzidine (DAB) as substrate (Vector Lab, California, USA). After images were captured, the number of positive stained cells was quantified as previously described.14 (link),17 (link) Briefly, five sequential sections from each sample were stained and for each section, five areas were measured.14 (link),17 (link)
6
Osteoclast Activity Quantification
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Cells were fixed with 4% formaldehyde for 10 min and then were incubated in 50 mM citrate buffer (50 mM citric acid and 50 mM sodium citrate (pH 4.5)) containing 5 mM 4-nitrophenylphosphate, and 10 mM sodium tartrate for 1 h. The reaction was terminated by adding 0.1 N NaOH. Absorption intensity was measured using a microplate reader at λ = 405 nm. Furthermore, cells were fixed with 4% formaldehyde and stained for 30 min with a commercially available TRAP kit (Sigma-Aldrich Chemical, St. Louis, MO, USA). TRAP-positive multinucleated osteoclasts were visualized under light microscopy (ECLIPSE Ni-U, Nikon, Tokyo, Japan).
7
Immunofluorescence and TRAP staining
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Cells were fixed with 4% paraformaldehyde-PBS for 15min. Following permeabilization and blocking with goat serum, cells were incubated with primary antibodies during 1 hour. Secondary antibodies used were conjugated with Alexa 488 and Alexa 555 from Jackson Immunoresearch (Interchim, Montluçon, France). Samples were mounted using Mowiol 4–88 reagent (Sigma Aldrich) supplemented with DAPI (Life technologies, St Aubin, France) and were analyzed using an upright Axioimager M2 microscope (Carl Zeiss SAS, Le Pecq, France.).
For paraffin-embedded tissues, sections were prepared and immunostained following deparaffinization and hydration. TRAP stainings were performed using the TRAP kit from Sigma Aldrich and according to the manufacturer’s instructions.
For paraffin-embedded tissues, sections were prepared and immunostained following deparaffinization and hydration. TRAP stainings were performed using the TRAP kit from Sigma Aldrich and according to the manufacturer’s instructions.
8
Histological and Immunohistochemical Analysis of Mouse Maxillas
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Mouse maxillas were fixed in paraformaldehyde, decalcified, embedded in paraffin, sectioned (5 μm thickness) and processed for hematoxylin and eosin staining or immunohistochemistry. Sections were briefly incubated with the following primary antibodies: goat anti-COX2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-NAMPT (AdipoGen) and rabbit anti-MMP3 (Abcam, Cambridge, UK). To detect osteoclasts, a leukocyte acid phosphatase (tartrate-resistant acid phosphatase; TRAP) kit (Sigma-Aldrich) was used. For double immunofluorescence labeling of human GF, cells were cultured on glass coverslips and permeabilized with 0.1% Triton X-100. Cells were incubated for 1 h with primary antibodies followed by 1 h with an Alexa 488- or Alexa 594-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). Images were acquired using a fluorescence microscope (Carl Zeiss, Cambridge, Jena, Germany) and were analyzed by counting positively stained cells using ImageJ software.
9
TRAP Staining Protocol for Histology
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Serial sections were stained for tartrate-resistant acid phosphate (TRAP) with commercial TRAP kit (Sigma, St Louis, USA). The sections were incubated in the incubation solution made up of Fast Garnet GBC Base Solution, sodium nitrate solution, naphthol AS-BI phosphoric acid, acetate solution, tartrate solution and deionized water for 60 min at 37 °C.
10
Osteoclast Differentiation and Characterization
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Total BM cells were seeded in 96 well plates at the density of 125,000 cells/well and differentiated in OC according to the protocol mentioned above up to 5 days. After day 5, media from the cultured cells was collected and used for the measurement of TRAcP activity according to the manufacture’s protocol47 (link),48 (link). Then, the cells were fixed with 10% formalin and staining for TRAcP using TRAP kit (Sigma, cat. n. 387A-1KT). Mature OCs were defined as multi-nuclei cells with three or more nuclei. TRAcP activity was normalized to the number of OCs.
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