Immunohistochemical staining was performed to detect DLX5 and BMP-2 using the streptavidin-peroxidase complex method. Cartilage tissues were fixed in neutral-buffered formalin, decalcified with EDTA, paraffin-embedded, sectioned into 6 μm sections, and mounted onto glass slides. The sections were then dewaxed with xylene and rehydrated with graded alcohol. Sections were incubated in citrate buffer for antigen retrieval. Hydrogen peroxide (3%) and BSA (5%) were used to block endogenous peroxidase and nonspecific binding of antibodies, respectively. The sections were incubated with anti-DLX5 (Abcam, ab109737) and anti-BMP-2 (Abcam, ab214821) primary antibodies at 4°C overnight and followed by incubation with a secondary antibody for 2 hours at room temperature. The streptavidin-peroxidase complex reagent and DAB solution were applied for visualization of expressions of DLX5 and BMP-2.
Ab109737
Manufactured by Abcam
Sourced in China, Canada
Ab109737 is an antibody product manufactured by Abcam. It is a monoclonal antibody that recognizes a specific target protein. The core function of this product is to bind and detect the target protein in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab214821, by Abcam (1 mentions)
Ab192256, by Abcam (1 mentions)
Ab209484, by Abcam (1 mentions)
Ab223692, by Abcam (1 mentions)
Ab185966, by Abcam (1 mentions)
Ab182563, by Abcam (1 mentions)
Ab264077, by Abcam (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
Nupage novex system, by Thermo Fisher Scientific (1 mentions)
Ecl system, by Cytiva (1 mentions)
4 protocols using ab109737
1
Immunohistochemical Analysis of DLX5 and BMP-2
2
Osteogenic Differentiation Markers in C3H10T1/2 Cells
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After 24 h culture, C3H10T1/2 (6 groups) in all six groups were harvested and extracted the total RNA and proteins. The genetic and proteic expression levels of runt-related transcription factor 2 (Runx2), osterix (Osx) and distal-less homeobox 5 (Dlx5) were detected in this work. The specific primers were shown in Table 1 . The procedures of qRT-PCR and Western blot are same as described before. Primary antibodies involved in this work were rabbit anti-Runx2 (Abcam, ab192256), rabbit anti-Osx (Abcam, ab209484) and rabbit anti-Dlx5 (Abcam, ab109737). The following second antibody was goat anti-rabbit (Abcam, ab223692).
3
Protein Expression Analysis in Cartilage Cells
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The cells in the culture were lysed using the RIPA buffer (Pierce, Rockford, IL, USA) with a protease inhibitor cocktail (Pierce). Total protein was separated by SDS-PAGE electrophoresis using the Novex Nu PAGE system (Invitrogen) and transferred to 0.45-μm PVDF membranes. Membranes were blocked in TBS-T buffer containing 5% nonfat milk for 1 h and incubated overnight at 4 °C with the following primary antibodies: anti-Collagen X (1:1,000, Abcam, #ab182563), anti-DLX5 (1:1,000, Abcam, #ab109737), anti-RUNX2 (1:1,000, Abcam, #ab264077), anti-SOX9 (1:1,000, Abcam, #ab185966), anti-MMP13 (1:1,000, Abcam, #ab51072), and anti-β-ACTIN (1:1,000, Beyotime, China, #AF0003) was used as the internal control. Then the membranes were washed using TBST and hybridized with the horseradish peroxidase (HRP)-linked antibody goat anti-rabbit IgG (1:4,000, Beyotime) or goat anti-mouse IgG for 1 h. Signal detection was carried out with an ECL system (Amersham Pharmacia, Piscataway, NJ, USA).
4
OA Mouse Model Development and Evaluation
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Animal experiments were performed under the approval of Animal Experimentation of Jiangsu University and according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. C57BL/6 male mice at 4 weeks old were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (Jiangsu, China). After one-week of adaptive feeding, the medial meniscal ligament (MMLT) in the right knee joint was severed to establish the OA model.42 (link),43 (link) For the sham group, a medial incision was operated on the knee joint without MMLT transection. After surgery, the mice were allowed to move and have access to a normal diet and tap water ad libitum. Six weeks after surgery, the mice were euthanized by continuous CO2 inhalation, the right joints were harvested and fixed in 4% paraformaldehyde for 48 h. Whole joints were decalcified in 10% EDTA with rotation at room temperature for 2 weeks, then specimens were embedded in paraffin, and 5-μm frontal sections were taken and subjected to hematoxylin and eosin and safranin O-fast green staining. For immunohistochemistry analysis, sections were incubated with primary antibodies against type X collagen (COL10; ABclonal, Wuhan, China, catalog# A6889; dilution: 1:300) and DLX5 (Abcam, Toronto, ON, Canada, catalog# ab109737; dilution: 1:100).
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