Optimized protocols for in situ hybridization, immunohistochemistry, and immunofluorescence on skeletal tissue

Dou, Zelong; Chau, Michael; Muder, Daniel; Vedung, Torbjörn; Nilsson, Ola

doi:10.1016/j.acthis.2021.151747

Cited by 5 publications

(5 citation statements)

References 23 publications

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“…After cleaning, pour in 10% EDTA decalcification solution (Solarbio, Beijing, China). It was estimated that decalcification will last for 4 weeks ( Savi et al, 2017 ; Bogoevski et al, 2019 ; Dou et al, 2021 ; Alaeddini et al, 2022 ). When the samples was elastic, it indicated that decalcification was complete, and the samples was stored in 75% alcohol.…”

Section: Methodsmentioning

confidence: 99%

Rat bone marrow mesenchymal stem cells induced by rrPDGF-BB promotes bone regeneration during distraction osteogenesis

Zhang²,

Zhang³

et al. 2023

Front. Bioeng. Biotechnol.

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Background: In the clinical treatment of large bone defects, distraction osteogenesis can be used. However, some patients may suffer from poor bone regeneration, or even delayed healing or non-union. Problems with the aggregation and proliferation of primary osteoblasts, or problems with the differentiation of primary osteoblasts will lead to poor bone regeneration. Therefore, supplementing exogenous primary osteoblasts and growth factors when using distraction osteogenesis may be a treatment plan with great potential.Methods: Bone marrow mesenchymal stem cells (BMSCs) were extracted from rats and cultured. Subsequently, Recombinant Rat Platelet-derived Growth Factor BB (rrPDGF-BB) was used to induce bone marrow mesenchymal stem cells. At the same time, male adult rats were selected to make the right femoral distraction osteogenesis model. During the mineralization period, phosphate buffer salt solution (control group), non-induction bone marrow mesenchymal stem cells (group 1) and recombinant rat platelet-derived growth factor BB intervened bone marrow mesenchymal stem cells (group 2) were injected into the distraction areas of each group. Then, the experimental results were evaluated with imaging and histology. Statistical analysis of the data showed that the difference was statistically significant if p < 0.05.Results: After intervention with recombinant rat platelet-derived growth factor BB on bone marrow mesenchymal stem cells, the cell morphology changed into a thin strip. After the cells were injected in the mineralization period, the samples showed that the callus in group 2 had greater hardness and the color close to the normal bone tissue; X-ray examination showed that there were more new callus in the distraction space of group 2; Micro-CT examination showed that there were more new bone tissues in group 2; Micro-CT data at week eight showed that the tissue volume, bone volume, percent bone volume, bone trabecular thickness, bone trabecular number and bone mineral density in group 2 were the largest, and the bone trabecular separation in group 2 was the smallest. There was a statistical difference between the groups (p < 0.05); HE staining confirmed that group 2 formed more blood vessels and chondrocytes earlier than the control group. At 8 weeks, the bone marrow cavity of group 2 was obvious, and some of them had been fused.Conclusion: The study confirmed that injecting bone marrow mesenchymal stem cellsBB into the distraction space of rats can promote the formation of new bone in the distraction area and promote the healing of distraction osteogenesis.

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Section: Methodsmentioning

confidence: 99%

Rat bone marrow mesenchymal stem cells induced by rrPDGF-BB promotes bone regeneration during distraction osteogenesis

Zhang²,

Zhang³

et al. 2023

Front. Bioeng. Biotechnol.

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show abstract

“…A magnification microscope (Zeiss X-Cite) was used to observe the stained cells at a 200× magnification [ 24 , 57 ]. Subsequently, ImageJ software (1.52a, Wayne Rasband, Bethesda, MD, USA) was used to process the fluorescent images [ 58 ].…”

Section: Methodsmentioning

confidence: 99%

Multiple Beneficial Effects of Aloesone from Aloe vera on LPS-Induced RAW264.7 Cells, Including the Inhibition of Oxidative Stress, Inflammation, M1 Polarization, and Apoptosis

Wang

Xiong

et al. 2023

Molecules

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Aloesone is a major metabolic compound in Aloe vera, which has been widely used as a food source and therapeutic agent in several countries. Our recent study demonstrated that aloesone has anti-epileptic effects on glutamate-induced neuronal injury by suppressing the production of reactive oxygen species (ROS). Unless ROS are naturally neutralized by the endogenous antioxidant system, they lead to the activation of inflammation, polarization, and apoptosis. This study aimed to identify the multiple beneficial effects of aloesone and explore its molecular mechanism in macrophages. Hence, the murine macrophage cell line RAW264.7 was pretreated with aloesone and then exposed to lipopolysaccharides (LPS). The results demonstrated that aloesone, within a dosage range of 0.1–100 µM, dramatically decreased the LPS-induced elevation of ROS production, reduced nitric oxide (NO) release, inhibited the M1 polarization of RAW264.7 cells, and prevented cells from entering the LPS-induced early and late phases of apoptosis in a dose-dependent manner. Simultaneously, aloesone significantly decreased the mRNA expression of inflammation-related genes (iNOS, IL-1ꞵ, TNF-α) and increased the expression of antioxidant enzymes (Gpx-1 and SOD-1). The core genes HSP90AA1, Stat3, Mapk1, mTOR, Fyn, Ptk2b, and Lck were closely related to these beneficial effects of aloesone. Furthermore, immunofluorescence staining and flow cytometry data confirmed that aloesone significantly repressed the activation of mTOR, p-mTOR, and HIF-1α induced by LPS and inhibited the protein expression of TLR4, which is the target of LPS. In conclusion, aloesone demonstrated multiple protective effects against LPS-induced oxidative stress, inflammation, M1 polarization, and apoptosis in macrophages, suggesting its potential as a prodrug.

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“…Immunohistochemistry was conducted as previously described 32 , 33 . Briefly, paraffin-embedded tissue sections were baked at 65 °C for 1 h, deparaffinized in xylene, rehydrated through an ethanol series (100%, 100%, 95%, and 70%), and rinsed in PBS.…”

Section: Methodsmentioning

confidence: 99%

“…After the preparation of DIG-labeled riboprobes, non-radioactive digoxigenin in situ hybridization was performed as previously described with slight modifications 33 , 35 . The detailed protocol is available upon request.…”

Section: Methodsmentioning

confidence: 99%

The synovial microenvironment suppresses chondrocyte hypertrophy and promotes articular chondrocyte differentiation

et al. 2022

Self Cite

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During the development of the appendicular skeleton, the cartilaginous templates undergo hypertrophic differentiation and remodels into bone, except for the cartilage most adjacent to joint cavities where hypertrophic differentiation and endochondral bone formation are prevented, and chondrocytes instead form articular cartilage. The mechanisms that prevent hypertrophic differentiation and endochondral bone formation of the articular cartilage have not been elucidated. To explore the role of the synovial microenvironment in chondrocyte differentiation, osteochondral allografts consisting of articular cartilage, epiphyseal bone, and growth plate cartilage from distal femoral epiphyses of inbred Lewis rats expressing enhanced green fluorescent protein from a ubiquitous promoter were transplanted either in inverted or original (control) orientation to matching sites in wildtype littermates, thereby allowing for tracing of transplanted cells and their progenies. We found that no hypertrophic differentiation occurred in the growth plate cartilage ectopically placed at the joint surface. Instead, the transplanted growth plate cartilage, with time, remodeled into articular cartilage. This finding suggests that the microenvironment at the articular surface inhibits hypertrophic differentiation and supports articular cartilage formation. To explore this hypothesis, rat chondrocyte pellets were cultured with and without synoviocyte-conditioned media. Consistent with the hypothesis, hypertrophic differentiation was inhibited and expression of the articular surface marker lubricin ( Prg4 ) was dramatically induced when chondrocyte pellets were exposed to synovium- or synoviocyte-conditioned media, but not to chondrocyte- or osteoblast-conditioned media. Taken together, we present evidence for a novel mechanism by which synoviocytes, through the secretion of a factor or factors, act directly on chondrocytes to inhibit hypertrophic differentiation and endochondral bone formation and promote articular cartilage formation. This mechanism may have important implications for articular cartilage development, maintenance, and regeneration.

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Optimized protocols for in situ hybridization, immunohistochemistry, and immunofluorescence on skeletal tissue

Cited by 5 publications

References 23 publications

Rat bone marrow mesenchymal stem cells induced by rrPDGF-BB promotes bone regeneration during distraction osteogenesis

Rat bone marrow mesenchymal stem cells induced by rrPDGF-BB promotes bone regeneration during distraction osteogenesis

Multiple Beneficial Effects of Aloesone from Aloe vera on LPS-Induced RAW264.7 Cells, Including the Inhibition of Oxidative Stress, Inflammation, M1 Polarization, and Apoptosis

The synovial microenvironment suppresses chondrocyte hypertrophy and promotes articular chondrocyte differentiation

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