Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.
Skeletal bone is the main load-bearing organ of the human body and is under pressure from both the internal and external environment during walking and exercise1. Throughout one’s life, bones continuously go through self-renewal, which is balanced by osteoblasts and osteoclasts. The process of osteoclasts clearing old bones and osteoblasts forming new bone maintains the homeostasis and mechanical function of the skeletal system2. Disturbance in the balance may induce bone metabolic diseases, such as osteoporosis. Osteoporosis, which is caused by excess osteoclastic activity, is globally prevalent and causes substantial economic losses to society2,3,4. According to the limited number of drugs available for osteoporosis treatment and their risk of adverse effects4, it is important to unveil the details of osteoclast formation and activity.
Osteoclasts derived from the monocyte/macrophage hematopoietic lineage have multiple nuclei (may have 2 to 50 nuclei) and are large (usually greater than 100 μm in diameter)2. Although the exploration of mechanisms and the screening of drugs for osteoclastic disorders have been widely improved via in vitro osteoclast culture, the complicated organic reactions make in vivo evidence indispensable for the targeted therapy. Due to genetic and pathophysiological similarities between mice and humans, genetically engineered mouse models are commonly used for studying the mechanisms and the pharmaceutical treatments of human disease in vivo6. The Cre-loxP system is a widely-used technology for mouse gene editing and has enabled researchers to investigate gene functions in a tissue-/cell-specific manner5. Cathepsin K (CSTK) is a cysteine protease secreted by osteoclasts that can degrade bone collagen8. It is well accepted that CTSK is selectively expressed in mature osteoclasts; therefore, Ctsk-Cre mice are considered to be a useful tool for functional studies of osteoclasts and has been used6.
The signal transducer and activator of transcription (STAT) family is classical and highly significant in immunity and cancer progression and development7,8. Among seven STATs, STAT3 is reported to be the most relevant to bone homeostasis9,10. Several in vivo studies have reported that specific inactivation of STAT3 in osteoblasts decreases bone formation9,10. Nevertheless, solid evidence regarding the participation of STAT3 in osteoclast formation and bone metabolism in vivo is still limited. Recently, we provided in vivo evidence with an osteoclast-specific Stat3 deletion mouse model (Stat3fl/fl; Ctsk-Cre, hereafter called Stat3Ctsk) that STAT3 participates in osteoclast differentiation and bone metabolism11. In the present study, we describe the methods and protocols that we used to analyze the changes in bone mass, bone histomorphology, and bone anabolism and catabolism of the Stat3Ctsk mice in order to study the influence of osteoclast-specific STAT3 deletion on bone homeostasis.
All methods relating to the animals described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiaotong University School of Medicine.
1. Breeding of osteoclast specific Stat3 deletion mice
NOTE: Stat3fl/fl mice were obtained commercially. Ctsk-Cre mice were provided by S. Kato (University of Tokyo, Tokyo, Japan12). The mice were bred and maintained under specific pathogen-free (SPF) conditions in the institutional animal facility under standardized conditions.
- Pair a sexually mature male mouse with two female mice of the same age. After 18 days, check daily checks for newborns. Separate the pregnant female mice and keep it alone if needed. Change male mice among different breeding cages if the female mice were not pregnant within 1 month of the pairing.
- Cross Stat3fl/fl mice to Ctsk-Cre mice (F0). Clip the tails for genotyping and keep the male Stat3fl/+; Ctsk-Cre mice until they are sexually mature, which is around 6 weeks of age (F1). Use the following primers: Stat3 F-TTGACCTGTGCTCCTACAAAAA; Stat3 R-CCCTAGATTAGGCCAGCACA; Ctsk-cre F-GAACGCACTGATTTCGACCA; Ctsk-cre R-GCTAACCAGCGTTTTCGTTC.
- Cross 6-week-old male Stat3fl/+; Ctsk-Cre mice with female Stat3fl/fl mice. Clip the tails for genotyping and keep the male Stat3 fl/fl; Ctsk-Cre mice until they are sexually mature, which is around 6 weeks of age (F2).
- Cross 6-week-old male Stat3 fl/fl; Ctsk-Cre mice with female Stat3fl/fl mice (F3). Clip the tails for genotyping and replace the old breeding mice with younger mice in time (F3+N).
2. Specimen collection
- Euthanize six pairs of 8-week old male Stat3fl/fl and Stat3Ctsk littermate mice separately with carbon dioxide asphyxiation.
NOTE: The CO2 flow rate displaces 30% of the cage volume per minute (e.g., for 45 cm x 30 cm x 30 cm cage the CO2 flow rate is 40 L/min).
- Place the mice in a supine position. Dislocate the bilateral hip joints gently by hand. Use ophthalmic scissors to cut off the skin vertically from the distal tibia and then dissect the entire skin from the hind limb.
- Cut off the articular ligament of the right hip joint and knee joint with scissors to separate the hind limb. Cut the trochanter and the junction of the fibula and then immerse the hind limb in 4% paraformaldehyde. Keep the right hind limbs for step 3. Appropriately cut the bone at both ends to fully immerse and fix the bone marrow with 4% paraformaldehyde.
- Cut the articular ligament of the left hip joint and knee joint with scissors, gently remove the soft tissue, and carefully separate the tibia and femur. Immerse the tibia and femur separately in 75% ethanol. Keep the femora for step 4 and tibiae for step 6. Ensure to keep the trochanter intact.
3. Paraffin section preparation
- Fix the right hind limb in 4% paraformaldehyde at 4 °C for 48 h.
- Decalcify: Gently wash the specimens with 1x PBS for 10 min 3 times. Decalcify the specimens in 15% EDTA (150 g EDTA in 800 mL of ddH2O and 100 mL of 10x PBS) with an ultrasonic decalcifier for 3 to 4 weeks until the bones can be bent. Replace with fresh decalcifying fluid every other day.
- Gently wash the specimens 3x with 1x PBS and then immerse them in 75% ethanol at 4 °C overnight.
- Dehydrate: On the second day, sequentially immerse specimens in 95% ethanol, 100% ethanol, and xylene, each for 1 h twice.
- Immerse specimens in 1/2 xylene 1/2 paraffin for 30 min. Immerse the specimens in paraffin at 65 °C overnight.
- Embed: Select a suitable embedding tank for embedding. Place the tibia uniformly underneath. Place the femur and tibia at a 90° angle. After the paraffin has fully cooled and solidified, remove it from the embedding tank. Number the specimens and store them at -20 °C overnight.
- Cut 5 μm thick sections continuously using the microtome. Cut 20–40 sections. Spread the sections on 37 °C water, adhere them to microscope slides, and bake at 42 °C overnight.
4. Micro-CT scanning and analysis
- Scan the left femora with a micro-CT scanner. Resolution: 10 μm; Voltage: 70 kV; Current: 114 μA; Fliter: 0.5 mm Al; Rotation step: 0.5°.
- Reconstruct 3D images of the cortical bone and trabecular bone using the scanner’s supporting software following manufacturer’s instruction. ROIs are in a total 1 mm width of trabecular bone close to the distal growth plate and in a total 1 mm wide section of cortical bone in the middle of the femora.
- Calculate the quantitative microarchitecture parameters: bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th.), trabecular number (Tb.N.), trabecular separation (Tb.Sp.), and cortical bone thickness (Ct.Th.).
5. TRAP staining
- Bake the paraffin sections at 65 °C for 30 min.
- Dewax: Immerse the sections in xylene for 10 min. Perform this step 3x with fresh xylene.
- Rehydrate: Immerse the sections sequentially in 100% ethanol, 95% ethanol, 70% ethanol, and ddH2O, each for 5 min twice.
- Prepare the staining solution using the TRAP staining kit following the manufacturer’s instructions and warm to 37 °C.
NOTE: TRAP staining solution should be freshly prepared immediately before every assay.
- Add 50–100 μL staining solution to each sample and incubate in a 37 °C humid chamber for 20–30 min. Check the staining status of the osteoclasts under a light microscope every 5 min until red multinucleated osteoclasts can be seen. End the reaction with ddH2O.
- Counterstain in hematoxylin solution for 30 s. Create a stable blue color by immersion in 1% ammonia solution for 1 min. Rinse in slowly running tap water.
- Mount the sections with coverslips using neutral balsam and dry overnight.
- Capture 3–5 fields of interest by a microscope. Analyze the trabecular perimeter by Image J: measure the length of scale bar (Ls) using the ‘straight line’ tool as L1, then measure the length of trabecular perimeter using the ‘segmented line’ tool as L2, the physical length (Lp)= Ls*L2 /L1). Count the number of TRAP-positive cells with more than three nuclei.
6. Calcein and alizarin red double labeling
- Specimen preparation: Intraperitoneally inject 20 mg/kg calcein (1 mg/mL in 2% NaHCO3 solution) on day 0, and 25 mg/kg alizarin red S (AL, 2 mg/mL in H2O) on day 4. Sacrifice mice on day 7. Carefully disassociate the tibiae and fix in 4% paraformaldehyde at 4 °C for 48 h.
- Dehydrate: After fixation, gently wash the tibiae 3x with 1x PBS. Sequentially immerse the specimens in 95% ethanol, 100% ethanol, and xylene for 5 min twice separately.
- Immerse the specimens in acetone for 12 h, in 1/2 acetone 1/2 resin for 2 h, and in pure resin in a drying oven overnight.
NOTE: The resin was prepared by commercially available resin (e.g., Embed 812 Resin) according to the manufacturer’s instructions.
- Embed: Add pure resin into a suitable silica gel embedding tank and place the specimens gently to avoid bubbles. Polymerize the resin in a drying oven at 60 °C for 48 h.
- Cut the specimens into 5 μm thick sections continuously with a rotary microtome. Store the rest of the samples with desiccant at room temperature.
- Adhere the sections with tweezers in a drop of 75% alcohol. Mount the sections with coverslips using neutral balsam. Capture red and green fluorescence labeling with a fluorescence microscope.
- Measure the width between two labeling lines (Ir.L.Wi), single-labeled trabecular perimeter (sL.Pm), double-labeled trabecular perimeter (dL.Pm), and total trabecular perimeter (Tb.Pm). Calculate the mineral apposition rate (MAR) and bone formation rate (BFR/BS). MAR = Ir.L.Wi/interval days. BFR/BS= (dL.Pm±sL.Pm/2)*MAR/Tb.Pm*100 %.
Using the present protocol, osteoclast specific Stat3 deletion mice were generated to study the influence of STAT3 deletion on osteoclast differentiation. Stat3Ctsk mice and their wildtype (WT) littermates were bred and kept after genotyping. Bone marrow macrophages were isolated and cultured into osteoclasts, and STAT3 deletion in Stat3Ctsk mice was demonstrated (Figure 1).
Femora reconstruction and quantitative analysis by micro-CT indicated that the bone mass of the Stat3Ctsk mice was increased compared with WT mice (Figure 2).
Histomorphology of the femora from WT and Stat3Ctsk mice was examined via H&E staining (Figure 3).
Osteoclastogenic activity in the mice was detected by TRAP staining. Osteoclasts were TRAP+ (wine red or purple) cells with multiple nuclei and huge size (Figure 4A). The number of TRAP+ osteoclasts was lower in the Stat3Ctsk mice compared with the WT mice (Figure 4B), which indicates that STAT3 deficiency impaired osteoclast formation.
Osteogenesis in mice is represented by the mineral apposition rate (MAR) and was measured by calcein and alizarin red double labeling (Figure 5). Calcein and alizarin red were sequentially intraperitoneally injected. Therefore, the area between the calcein (green) and alizarin red (red) fluorescence lines represents newly formed bone over four days. As shown in Figure 5, deleted STAT3 in osteoclasts did not influence bone anabolism.
Figure 1: Illustration of osteoclast specific Stat3 deletion mouse model generation and genetically engineered mice. (A) Schematic diagram of Stat3 deletion in cathepsin K (Ctsk)-expressing osteoclasts via the Cre-loxP system. (B) Breeding progress of osteoclast specific Stat3 deletion mice. Stat3fl/fl mice were crossed to Ctsk-Cre mice (F0) to generate heterozygous Stat3fl/+; Ctsk-Cre mice (F1). Male Stat3fl/+; Ctsk-Cre mice were kept and crossed to female Stat3fl/fl mice to generate homozygous Stat3fl/fl; Ctsk-Cre mice (F2). Male Stat3fl/fl; Ctsk-Cre mice were kept and crossed to female Stat3fl/fl mice to generate Stat3fl/fl; Ctsk-Cre mice and Stat3fl/fl mice. (C) Genotyping for various genotypes of mice. Stat3 mutant: 187 bp, Stat3 wildtype: 146 bp, Cre: 200 bp. (D) Western blot of STAT3 in osteoclasts cultured with bone marrow macrophages from Stat3fl/fl and Stat3Ctsk mice. Please click here to view a larger version of this figure.
Figure 2: Three-dimensional reconstruction and quantitative microarchitecture parameter analysis in femora of mice using micro-CT scanning. (A) Three-dimensionally reconstructed micro-CT images of trabecular bone of femora from 8-week-old WT and Stat3Ctsk mice. The region of interest (ROI) was in a total 1 mm width of trabecular bone close to the distal growth plate. (B) 3D reconstructed micro-CT images of cortical bone of femora from 8-week-old WT and Stat3Ctsk mice. ROI was in a total 1 mm wide section of cortical bone from the middle of the femora. (C–H) Quantitative microarchitecture parameters of micro-CT: bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th.), trabecular number (Tb.N.), trabecular separation (Tb.Sp.), and cortical bone thickness (Ct.Th.). Error bars represent the mean ± SD, n=4, *P<0.05. Please click here to view a larger version of this figure.
Figure 3: Histomorphology of femora from WT and Stat3Ctsk mice exhibited via H&E staining. M-shaped dotted curve indicates a cartilage layer. Relative high-power field of cortical bone is boxed with dotted lines and exhibited below. Relative high-power field of trabecular bone is circled with dotted lines and exhibited at the bottom. Please click here to view a larger version of this figure.
Figure 4: Osteoclastogenesis in femora of mice represented via TRAP staining. (A) TRAP staining of femora from 8-week-old WT and Stat3Ctsk mice. TRAP+ multinucleated osteoclasts are indicated by black triangles. Relative high-power field of osteoclasts circled with dotted lines are exhibited below, demonstrating multiple nuclei and a huge size. (B) The numbers of TRAP+ multinucleated osteoclasts were counted. Error bars represent the mean ± SD, n=5, *P<0.05. Please click here to view a larger version of this figure.
Figure 5: Osteogenesis in femora of mice represented via calcein and alizarin red double labeling. (A) Seven-week-old mice were sequentially injected with calcein on day 0, with alizarin red on day 4, and then sacrificed on day 7. The area between calcein (green) and alizarin red (red) fluorescence lines represent newly formed cortical bone over four days. (B) Mineral apposition rate (MAR) and bone formation rate (BFR/BS) calculated by the distance of the two lines represent the osteogenic activity of the cortical bone. (C) The area between the calcein (green) and alizarin red (red) fluorescence lines represent newly formed trabecular bone in four days. (D) MAR and BFR/BS represent the osteogenic activity of the trabecular bone. Error bars represent the mean ± SD, n=5, *P<0.05. Please click here to view a larger version of this figure.
Genetically engineered mouse models are commonly used for studying the mechanism and pharmaceutical treatment of human disease13. Ctsk-Cre mice have been widely used for functional studies of osteoclasts6. The present study described the protocols of the methods to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.
Histological analysis is the best intuitive method to detect bone metabolism. And the quality of the paraffin sections is the base of the histological analysis. Sufficient time for full fixation and decalcification of the bone tissue is extremely essential since bone tissues can be fragmented. Researchers focusing on femora and tibiae should place the femur and tibia at a 90° angle in the embedding step. In order to conduct reliable and high-quality histological analysis, we chose sagittal paraffin sections in which the cartilage layer was symmetrical and showed a clear M-shaped line in H&E staining, which represents the suitable angle and depth of those continuous sections. On the other hand, if you need to observe the knee joints and adjacent articular ligament, the femur and tibia should be placed at a 120° angle.
TRAP has served for decades as a biochemical marker for osteoclast function14, since it is predominantly expressed in osteoclasts15,16. Two substrates are usually used to assess acid phosphatase activity. Naphthol-ASBI phosphate (N-ASBI-P) is an excellent substrate for the osteoclast-specific TRAP isoform 5b. However, para-nitrophenyl phosphate (pNPP) can also be hydrolyzed by nontype 5 TRAPs17. Therefore, the naphthol-ASBI phosphoric acid-pararosaniline method for histochemical demonstration of TRAP is highly specific in tissue sections17,18,19. Macrophage phosphatase (acid phosphatase) has a pH optimum of 5.0–6.0, at which it is mostly tartrate resistant20. Thus, we recommend that in the protocol of TRAP staining assay, tissue sections re-stained by hematoxylin should not be decolored with hydrochloric acid. Importantly, although osteoblasts and osteocytes close to bone remodeling areas also express TRAP21, only huge TRAP-positive cells with more than three nuclei were considered as osteoclasts. In this study, Stat3Ctsk mice showed inhibited osteoclast activity (Figure 4).
Calcein is a fluorochrome (green fluorescence) that is widely used to indicate skeletal growth during calcification. It can bind to calcium and be incorporated into the newly formed calcium carbonate crystals22. Similarly, calcein, alizarin red (red fluorescence), and tetracycline (yellow fluorescence) were built into the bone to estimate growth from the time of exposure23,24. Many studies in the past have used single fluorochromes (usually calcein) to label bone, but observers can be easily confused between the newly formed bone and old bone, especially in irregular trabecular bone. Therefore, we suggest that researchers inject two different types of fluorochromes into mice to distinguish the newer bone from the older bone. According to our tests, calcein coupled with alizarin might achieve the most sufficient and enduring contrast. In this study, calcein-alizarin red labeling was used to detect the osteogenic rate of bone tissue and it turned out that osteoblast activity was not influenced in the Stat3Ctsk mice (Figure 5).
In short, to understand the critical genes controlling osteoclast activity, this protocol describes a canonical method to generate a transgenic mouse model. This protocol also describes some typical techniques for analyzing the skeletal phenotype. In this study, the deletion of STAT3 impaired osteoclast formation and increased bone mass. These techniques may be interesting for those who are new to skeletal tissue research. However, more characteristics of skeleton system are concerned for further study, such as mechanical property25. And we always need to keep eyes on the development of new techniques.
The authors have nothing to disclose.
We thank Prof. Weiguo Zou and S. Kato for reagents and mice and the members of the Zou laboratory for useful discussions. We also thank the Laboratory for Digitized Stomatology and Research Center for Craniofacial Anomalies of Shanghai Ninth People's Hospital for assistance. This work was supported in part by grants from the National Natural Science Foundation of China (NSFC) [81570950,81870740,81800949], Shanghai Summit & Plateau Disciplines, the SHIPM-mu fund from the Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine [JC201809], the Incentive Project of High-Level Innovation Team for Shanghai Jiao Tong University School of Medicine, the Cross-disciplinary Research Fund of Shanghai Ninth People's Hospital, Shanghai JiaoTong university School of Medicine [JYJC201902]. And L.J. is a scholar of the Outstanding Youth Medical Talents, Shanghai "Rising Stars of Medical Talent" Youth Development Program and the “Chen Xing” project from Shanghai Jiaotong University.
|4% Paraformaldehyde solution||Sangon biotech Co., Ltd.||E672002|
|Acetone||Shanghai Experimental Reagent Co., Ltd.||80000360|
|Ammonia solution||Shanghai Experimental Reagent Co., Ltd.|
|Ctsk-Cre mice||a gift from S. Kato, University of Tokyo, Tokyo, Japan|
|DDSA||Electron Microscopy Sciences||13710|
|DMP-30||Electron Microscopy Sciences||13600|
|EDTA||Shanghai Experimental Reagent Co., Ltd.||60-00-4|
|EMBED 812 RESIN||Electron Microscopy Sciences||14900|
|Hematoxylin solution||Beyotime Biotechanology||C0107|
|Micro-CT||Scanco Medical AG||μCT 80|
|NaHCO3||Shanghai Experimental Reagent Co., Ltd.||10018918|
|Neutral balsam||Sangon biotech Co., Ltd.||E675007|
|NMA||Electron Microscopy Sciences||19000|
|Paraffin||Sangon biotech Co., Ltd.||A601889|
|Stat3fl/fl mice||GemPharmatech Co., Ltd||D000527|
|TRAP staining kit||Sigma-Aldrich||387A|
|xylene||Shanghai Experimental Reagent Co., Ltd.||1330-20-7|
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