On day 36, mice were euthanized to collect ankle joints. The samples were fixed in 10% phosphate-buffered formaldehyde solution for 24 h, then decalcified in a 10% ethylenediaminetetraacetic acid solution for 1 month, dehydrated and embedded in paraffin. TGF-β expression in ankle joints was measured as previously described [29 (link)]. Tissue sections (thickness, 3 μm) were incubated with primary antibodies against TGF-β (Abcam, Cambridge, MA, USA) overnight at 4 °C. Subsequently, they were incubated with HRP-conjugated goat anti-rabbit IgG antibody (DAKO, Carpinteria, CA, USA) to detect immunoactivity, which was followed by detection using a DAB solution kit (DAKO). The counterstain was hematoxylin. Stained specimens were visualized under a virtual microscope (Axio Scan.Z1; Carl Zeiss, Heidenheim, Germany). Histological results were the average of scores from three independent observers blinded to the experimental conditions. TGF-β expression was analyzed Image J (NIH; National Institutes of Health).
Primary antibodies against tgf β
Manufactured by Abcam
Sourced in United States
Primary antibodies against TGF-β. These antibodies are used to detect and measure the presence of the transforming growth factor-beta (TGF-β) protein in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti rabbit igg antibody, by Agilent Technologies (1 mentions)
Safranin o, by Merck Group (1 mentions)
Fast green, by Merck Group (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
P smad2 3, by Abcam (1 mentions)
Envision staining kit, by Agilent Technologies (1 mentions)
2 protocols using primary antibodies against tgf β
1
TGF-β Expression in Murine Ankle Joints
2
Histological Analysis of HO Tissues
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Mice were sacri ced using carbon dioxide (CO 2 ) inhalation for an indicated period to be used for histological observations. The ankles with Achilles tendons were dissected and xed in 4% paraformaldehyde for 24 h. The HO tissues from patients were collected during surgical operation. Then, the tissues were decalci cated using 10% ethylenediaminetetracetic acid (EDTA) solution for 1 month.
The decalci ed tissues were processed using graded dehydration and were then embedded in para n. 5 μm thick histological sections were obtained using a microtome and were subsequently processed for staining.
For SOFG staining, the sections were stained with 0.1% Safranin-O and 0.02% Fast Green (Sigma-Aldrich, Oakville, ON, Canada) by following the manufacturer's instructions.
Immunohistochemical staining was carried out using primary antibodies against TGF-β, p-Smad2/3, CD68, CD31, and VEGF (Abcam) at a 1:500 dilution of the appropriate secondary antibody. Protein expression was visualized using a DakoCytomation Envision staining kit. The mean density of the positive area was measured using Image-Pro Plus 6.0 (IPP) image analysis software. Three random slides were selected, and the images of ve random elds were captured in each sample.
The decalci ed tissues were processed using graded dehydration and were then embedded in para n. 5 μm thick histological sections were obtained using a microtome and were subsequently processed for staining.
For SOFG staining, the sections were stained with 0.1% Safranin-O and 0.02% Fast Green (Sigma-Aldrich, Oakville, ON, Canada) by following the manufacturer's instructions.
Immunohistochemical staining was carried out using primary antibodies against TGF-β, p-Smad2/3, CD68, CD31, and VEGF (Abcam) at a 1:500 dilution of the appropriate secondary antibody. Protein expression was visualized using a DakoCytomation Envision staining kit. The mean density of the positive area was measured using Image-Pro Plus 6.0 (IPP) image analysis software. Three random slides were selected, and the images of ve random elds were captured in each sample.
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