387a 1kt

Manufactured by Merck Group

Sourced in United States

The 387A-1KT is a laboratory equipment product. It is designed for specific laboratory applications. The core function of this product is to provide a standardized tool for controlled testing and analysis procedures. No further details can be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Methyl green, by Merck Group (3 mentions) Eclipse e800 microscope, by Nikon (3 mentions) Fast green, by Merck Group (2 mentions) α mem, by Thermo Fisher Scientific (2 mentions) 2030 rotary microtome, by Reichert Technologies (2 mentions) Excesior tissue processor, by Thermo Fisher Scientific (2 mentions) Dp71 camera, by Olympus (2 mentions) 86r 1kt, by Merck Group (2 mentions) Bx51 microscope, by Olympus (2 mentions) Safranin o, by Merck Group (2 mentions) β catenin, by Cell Signaling Technology (1 mentions) Envision staining kit, by Agilent Technologies (1 mentions) Superfrost plus slide, by Thermo Fisher Scientific (1 mentions) Ckx41, by Olympus (1 mentions) Rmrankl, by R&D Systems (1 mentions) Rm2235 microtome, by Leica (1 mentions) Glutamax supplement, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Human csf1, by Sino Biological (1 mentions) Human csf 1, by Merck Group (1 mentions)

40 protocols using 387a 1kt

TRAP Staining Protocol for Callus Analysis

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Tartrate-resistant acid phosphatase (TRAP) staining for callus was performed, as previously described^{29 (link),30 (link)}. In brief, decalcified sections were first deparaffinized and preincubated. Subsequently, the slides were incubated for 45–60 min at 37 °C with TRAP staining solution, based on the manufacturer’s protocol (387A-1KT, Sigma-Aldrich, MO, USA). The slides were also counterstained with hematoxylin solution. For quantification, the OC surface was calculated using an image processing program (ImageJ software, National Institutes of Health, Bethesda, MD, USA).

Zhao S.J., Kong F.Q., Cai W., Xu T., Zhou Z.M., Wang Z.B., Xu A.D., Yang Y.Q., Chen J., Tang P.Y., Wang Q., Cheng L., Luo Y.J., Zhou Z., Li L.W., Huang Y.F., Zhao X., Yin G.Y., Xue M.X, & Fan J. (2018). GIT1 contributes to autophagy in osteoclast through disruption of the binding of Beclin1 and Bcl2 under starvation condition. Cell Death & Disease, 9(12), 1195.

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Analysis of Achilles Tendon Inflammation

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Mice were killed by carbon dioxide (CO₂) inhalation and perfusion fixed with 10% buffered formalin via the left ventricle for 5 minutes. Then, the ankles with Achilles tendons were dissected and fixed in 4% paraformaldehyde for 24 hours. All of these specimens were decalcified in a 10% EDTA solution for 1 month, embedded in paraffin and cut into 5‐μm sections for staining.
Trap staining was performed following the manufacturer's protocol (Sigma‐Aldrich, 387A‐1KT), followed by counterstaining with Methyl Green (Sigma‐Aldrich, M884).
Sections were stained with 0.1% Safranin O and 0.02% Fast Green (Sigma‐Aldrich) according to the manufacturer's instructions.
Immunohistochemical staining was carried out with primary antibodies against IL‐17, IL‐17R (Abcam, Cat No. ab11370) and β‐catenin (Cell Signaling Technology, Cat No. 4370) with a 1:1000 dilution of an appropriate secondary antibody. Protein expression was visualized with a DakoCytomation EnVision staining kit. The mean density of the positive area was measured by Image‐Pro Plus 6.0 (IPP) image analysis software. Three random slides were selected, and five random fields of images per sample were taken.

Tu B., Yu B., Wang W., Li J., Yuan F., Zhu J, & Fan C. (2021). Inhibition of IL‐17 prevents the progression of traumatic heterotopic ossification. Journal of Cellular and Molecular Medicine, 25(16), 7709-7719.

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Histological Analysis of Decalcified Mouse Tissues

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Histology and tissue preparation were performed as described previously.^{10 (link)} Briefly, mice were euthanized, skinned and fixed in 4% paraformaldehyde overnight. The samples were then dehydrated in ethanol solution and decalcified in 10% EDTA for 2–4 weeks. For paraffin sections, samples were dehydrated in ethanol, cleared in xylene, embedded in paraffin, sectioned with a 5 μm Leica microtome and mounted on Superfrost Plus slides (Fisher). For frozen sections, decalcified samples were infiltrated with 30% sucrose, embedded in OCT, and sectioned at 8 μm by a freezing microtome. H&E (HE) staining and safranin O (SO) staining were performed as described previously.^{10 (link)} We used a commercial kit (Sigma-Aldrich, 387A-1KT) to perform tartrate-resistant acid phosphatase (TRAP) staining according to the manufacturer’s instructions.

Zhang Y., Zuo T., McVicar A., Yang H.L., Li Y.P, & Chen W. (2022). Runx1 is a key regulator of articular cartilage homeostasis by orchestrating YAP, TGFβ, and Wnt signaling in articular cartilage formation and osteoarthritis. Bone Research, 10, 63.

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Decalcification and TRAP Staining

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Tissues were fixed in 4% paraformaldehyde for 48 h and incubated in 15% DEPC-EDTA (PH7.8) for decalcification^{49 (link)}. Then specimens were embedded in paraffin and sectioned at 6 μm. Tissue sections were used for TRAP staining according to the manufacturer’s instructions (Sigma, 387A-1KT). Images were captured using a microscope (Olympus BX51, Tokyo, Japan).

Wang L., You X., Lotinun S., Zhang L., Wu N, & Zou W. (2020). Mechanical sensing protein PIEZO1 regulates bone homeostasis via osteoblast-osteoclast crosstalk. Nature Communications, 11, 282.

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Histological Evaluation of Tissue Samples

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For routine histology, H&E, Safranin-O, and tartrate-resistant acidic phosphatase (TRAP) stainings, tissue was fixed in 4% PFA/PBS and processed as previously reported.^{41 (link)} TRAP staining was performed according to the manufacturer’s indications (Sigma-Aldrich, 387A-1KT). In situ hybridization (ISH) analysis was performed using complementary ³⁵S-labeled riboprobes as previously described.^{41 (link)} H&E-, Safranin-O-, and TRAP-stained sections were used for static histomorphometry analysis. All images were acquired with an Eclipse E800 microscope (Nikon).

Merceron C., Ranganathan K., Wang E., Tata Z., Makkapati S., Khan M.P., Mangiavini L., Yao A.Q., Castellini L., Levi B., Giaccia A.J, & Schipani E. (2019). Hypoxia-inducible factor 2α is a negative regulator of osteoblastogenesis and bone mass accrual. Bone Research, 7, 7.

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TRAP Staining for Osteoclast Differentiation

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TRAP staining was performed as per the manufacturer’s protocol. Briefly, cells of different passages were seeded at a concentration of 1 × 10⁴ cells/mL in 96-well transparent plate and incubated for 24 h, followed by changing of the media with αMEM (Gibco) with or without 100 ng/mL recombinant mouse RANKL (rmRANKL, R&D Systems, Minneapolis, MN, USA) for five days. Media was renewed after three days. Cells were fixed with 10% formalin neutral buffer solution after culture for a total of five days with differentiation medium (RANKL containing medium). Staining of the cells was completed using a TRAP staining kit (387A-1KT, Sigma-Aldrich, St. Louis, MO, USA). A light microscope (Olympus CKX 41, Tokyo, Japan) was used to count the nuclei and cells. Multinucleated cells having ≥3 nuclei were regarded as osteoclast cells [11 ,12 ,13 ,14 ,15 (link)].

Lucy T.T., Mamun-Or-Rashid A.N., Yagi M, & Yonei Y. (2022). Serial Passaging of RAW 264.7 Cells Modulates Intracellular AGE Formation and Downregulates RANKL-Induced In Vitro Osteoclastogenesis. International Journal of Molecular Sciences, 23(4), 2371.

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Histomorphometric Analysis of Tibial Bone

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Tibias were fixed in 10% formalin for 24 hours then changed to 70% Ethanol. Fixed samples were processed on an automated Thermo Electron Excesior tissue processor for dehydration, clearing, and infiltration using a routine overnight processing schedule. Samples were embedded in Surgipath-embedding paraffin on a Sakura Tissue Teck II-embedding center. Paraffin blocks were sectioned at 5 μm on a Reichert Jung 2030 rotary microtome. Slides were stained for TRAP activity and counterstained with hematoxylin according to manufacturer’s protocol (387A-1KT, Sigma, St. Louis, MO). Slides were photographed in 5 sections per slide at 25x magnification for osteoblast and osteoclast counts and at 10x magnification for adipocytes. Image Pro-plus software was used in analysis of slide images. In the tibia trabecular region, ranging from the growth plate to 2mm toward the diaphysis, osteoblast and osteoclast surface area was measured and expressed as a percentage of total bone surface. Similarly, adipocytes greater than 30 μm in size were counted in the same area and expressed as the number per μm of marrow area. Histological analyses and measurements were performed in a blinded manner to treatment groups.

McCabe L.R., Irwin R., Tekalur A., Evans C., Schepper J., Parameswaran N, & Ciancio M. (2018). Exercise prevents high fat diet-induced bone loss, marrow adiposity and dysbiosis in male mice. Bone, 118, 20-31.

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Histological Analysis of Decalcified Bone

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Freshly dissected bones were fixed in 4% PFA for 48 h and incubated in 15% EDTA (pH 7.8) for decalcification. Specimens were then embedded in paraffin and sectioned at 7 μm using a Leica RM2235 microtome. Next, the paraffin sections were dewaxed, hydrated and stained with SO. The tissue sections were subjected to TRAP staining according to the manufacturer’s instructions (Sigma, 387A-1KT). Images were captured using an upright microscope (Olympus BX53).

Yang R., Cao D., Suo J., Zhang L., Mo C., Wang M., Niu N., Yue R, & Zou W. (2023). Premature aging of skeletal stem/progenitor cells rather than osteoblasts causes bone loss with decreased mechanosensation. Bone Research, 11, 35.

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Osteoclastogenesis from Human PBMCs

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Human PBMCs were isolated from healthy donors by Ficoll gradient centrifugation. Written informed consent was obtained from all donors. PBMCs were cultured in ɑ-MEM with 10% FBS (Sigma), 1% GlutaMAX Supplement (Thermo Fisher) and 30 ng ml⁻¹ human CSF-1 (Sino Biological, 11792-H08Y) and incubated in a humidified atmosphere at 37 °C and 5% CO₂. For osteoclastogenesis, 5 × 10⁵ PBMCs were seeded in a 12-well plate, after 48 h, cells were stimulated with 50 ng ml⁻¹ human RANKL (R&D, 6449–TEC-010) and 30 ng ml⁻¹ human CSF-1 for 10–14 d. Medium was changed every 2 d. Osteoclasts were fixed and stained using the TRAP staining kit (Sigma-Aldrich, 387A-1KT).

Lin L., Guo Z., He E., Long X., Wang D., Zhang Y., Guo W., Wei Q., He W., Wu W., Li J., Wo L., Hong D., Zheng J., He M, & Zhao Q. (2023). SIRT2 regulates extracellular vesicle-mediated liver–bone communication. Nature Metabolism, 5(5), 821-841.

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Quantification of Osteoclast and Osteoblast

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The paraffinized pathological sections were dewaxed in xylene and a graded ethanol series. TRAP staining was performed using a kit according to the manufacturer's instructions (387A-1KT; Sigma-Aldrich, St. Louis, MO, USA). ALP staining was performed using a kit according to the manufacturer's instructions (86R-1KT; Sigma-Aldrich, St. Louis, MO, USA). Osteoclast and osteoblast numbers were quantitated in the four different regions, and expressed as cells/mm2. Values were calculated for five nonconsecutive sections per region. Images were obtained with the aid of an Olympus BX51 microscope equipped with a DP71 camera (Olympus, Tokyo, Japan).

Wang C., Meng H., Wang Y., Zhao B., Zhao C., Sun W., Zhu Y., Han B., Yuan X., Liu R., Wang X., Wang A., Guo Q., Peng J, & Lu S. (2018). Analysis of early stage osteonecrosis of the human femoral head and the mechanism of femoral head collapse. International Journal of Biological Sciences, 14(2), 156-164.

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