Paraffin-embedded heads were sectioned at a thickness of 8 µm for immunohistochemistry. Subsequent to blocking with goat serum (1:10; Invitrogen Life Technologies, Carlsbad, CA, USA) and incubation for 15 min at room temperature, the sections were incubated with polyclonal antibodies against runt-related transcription factor 2 (Runx2; 1:1,000; ab76956), sex determining region Y-box 9 (Sox9; 1:500; ab26414), collagen type I (col I) (1:500; ab34710), collagen type II (col II) (1:200; ab53047), aggrecan (1:500; ab36861), matrix metalloproteinase 9 (MMP9) (1:300; ab38898), MMP13 (1:50; ab75606) and Ihh (1:200; ab39634) from Abcam (Cambridge, MA, USA) overnight at 4°C. The slides were then washed three times with PBS and were incubated with a biotinylated horseradish peroxidase goat anti-rabbit secondary antibody (1:1,000 dilution; Invitrogen Life Technologies) for 20 min at 37°C. The slides were then washed three times with the secondary antibody using PBS. Immunolabelling was visualized with 0.05% diaminobenzidine (Invitrogen Life Technologies) in PBS for 5 min at room temperature, and the slides were then rinsed for 10 min under running tap water. The TMJ immunohistochemical staining was analyzed under the Olympus BH-2 light microscope.
Ab39634
Manufactured by Abcam
Sourced in United States
Ab39634 is a laboratory product offered by Abcam. It is a piece of equipment designed for use in scientific research and analysis. The core function of this product is to facilitate the measurement and examination of samples within a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Ab88522, by Abcam (2 mentions)
Fluoroshield with dapi histology mounting medium, by Merck Group (2 mentions)
Proteinase k, by Promega (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Ab36861, by Abcam (1 mentions)
Ab26414, by Abcam (1 mentions)
Ab75606, by Abcam (1 mentions)
Ab76956, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Ab38898, by Abcam (1 mentions)
Bh 2 light microscope, by Olympus (1 mentions)
Ab23981, by Abcam (1 mentions)
Light microscopy, by Leica (1 mentions)
Mmp 13, by Santa Cruz Biotechnology (1 mentions)
Sox9 sc 166505, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Ab21286, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Sc 13119, by Santa Cruz Biotechnology (1 mentions)
6 protocols using ab39634
1
Immunohistochemical Analysis of TMJ Tissues
2
Chondrocyte Protein Expression Analysis
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Immunohistochemical (IHC) staining was performed to analyze the proteins expressed by chondrocyte-related genes. Tissue sections were processed for antigen retrieval by digestion with 0.05% trypsin at 37°C for 15 minutes. Tissue sections were incubated with primary antibodies to Sox9 (sc-166505) (Santa Cruz, USA), collagen type II alpha 1 (col2a1) (sc-517571) (Santa Cruz, USA), collagen type X (ColX) (ab58632) (Abcam, Cambridge, MA, USA), and matrix metalloproteinase 13 (MMP13) (sc-101564) (Santa Cruz, USA) overnight at 4°C. Then, sections were incubated with the secondary antibodies for 1 hour and developed in 3,3′-diaminobenzidine (DAB) chromogen (brown) (Invitrogen, USA). The sections were counterstained with hematoxylin, and observed by light microscopy (Leica, Germany).
For the immunofluorescence (IF) staining, the sections were stained with primary antibodies against runt-related transcription factor (RUNX2) (ab23981) (Abcam, Cambridge, MA, USA), Indian hedgehog (Ihh) (ab39634) (Abcam, Cambridge, MA, USA) and parathyroid hormone-related protein (PHrP) (sc-12722) (Santa Cruz, USA). Imaging was performed using a Leica fluorescence microscope. Quantification of the positively-stained cells was performed by microscopy.
For the immunofluorescence (IF) staining, the sections were stained with primary antibodies against runt-related transcription factor (RUNX2) (ab23981) (Abcam, Cambridge, MA, USA), Indian hedgehog (Ihh) (ab39634) (Abcam, Cambridge, MA, USA) and parathyroid hormone-related protein (PHrP) (sc-12722) (Santa Cruz, USA). Imaging was performed using a Leica fluorescence microscope. Quantification of the positively-stained cells was performed by microscopy.
3
Proteomic Analysis of Mandibular Explant
4
Bone Tissue Protein Immunoblotting
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Proteins were extracted from bone tissue, and immunoblotting was carried out as we described previously 31 (link). Immunoblotting was performed with the primary antibodies against Shh (ab135240; Abcam, Cambridge, MA, USA), Bmi1 (10832-1-AP; Proteintech, USA), Gli1 (ab49314; Abcam, Cambridge, MA, USA), Gli2 (18989-1-AP; Proteintech, USA), Gli3 (ab69838; Abcam, Cambridge, MA, USA), Ihh (ab39634; Abcam, Cambridge, MA, USA), Ptch1 (ab53715; Abcam, Cambridge, MA, USA), Sufu (#C54G2; Cell Signaling Technology, Beverly, MA, USA) and Smo (ab236465,Abcam, Cambridge, MA, USA). The β‐actin (BS6007M; Bioworld Technology, Bloomington, MN, USA) was used as the control for total protein.
5
Immunofluorescence Assay for Cartilage
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Immunofluorescence was performed based on previously described protocols. 42 Briefly, 3 µm knee joint sections were deparafinized and rehydrated. Antigen retrieval was performed by incubation with 20 µg/ml proteinase K (Promega, USA) for 20 min. Tissue sections were incubated with 0.1 M glycine for autofluorescence removal, and blocked with 3% PBS-bovine serum albumin (BSA), 0.1% Triton X-100 (Sigma), 5% FBS. Then cartilage sections were incubated with the corresponding primary antibodies: rabbit polyclonal anti-IHH (1/100; ab39634, Abcam) and mouse polyclonal anti-Cyclooxygenase-2 (COX-2) (1/100; ab88522; Abcam). Secondary FITC and TRITC respectively antibodies were used for detection of positive fluorescence signal. Tissue sections were ultimately incubated with 0.1% Sudan Black in 70% ethanol and mounted with Fluoroshield with DAPI histology mounting medium (Sigma). Sections were photographed with a confocal microscope Leica TCS SP5 (Leica Microsystems, Madrid, Spain) at ×40 magnification.
6
Immunofluorescence of Knee Joint Sections
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Immunofluorescence was performed based on previously described protocols (60) . Briefly, 3 µm knee joint sections were deparafinized and rehydrated. Antigen retrieval was performed by incubation with 20 µg/mL proteinase K (Promega, USA) for 20 minutes. Tissue sections were incubated with 0.1 M glycine for autofluorescence removal, and blocked with 3% PBS-bovine serum albumin (BSA), 0.1% Triton X-100 (Sigma), 5% FBS. Then cartilage sections were incubated with the corresponding primary antibodies: rabbit polyclonal anti-IHH (1/100; ab39634, Abcam) and mouse polyclonal anti-Cyclooxygenase-2 (COX-2) (1/100; ab88522; Abcam). Secondary FITC and TRITC respectively antibodies were used for detection of positive fluorescence signal. Tissue sections were ultimately incubated with 0.1% Sudan Black in 70% ethanol and mounted with Fluoroshield with DAPI histology mounting medium (Sigma).
Sections were photographed with a MiCom fluorescence microscope equipped with ACT-1 software at ×40 magnification.
Sections were photographed with a MiCom fluorescence microscope equipped with ACT-1 software at ×40 magnification.
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