A total of 14 root tendons collected from 2 to 4 weeks were subjected to histological examination, fixed in 10% formalin and embedded in paraffin. The embedded tendons were cut into 4 μm cross-sectional sections and stained with hematoxylin and eosin (HE). An independent examiner completed the histological interpretation at magnifications of ×10 and ×20 and calculated the number and density of Fibroblast (Fb) displacements. In addition, a total of 49 serum samples from 7 blood sampling sites were subjected to Enzyme-linked Immunosorbent Assay (ELISA) to test for the pro-inflammatory factor-rat interleukin IL-1β and the anti-inflammatory factor-growth transforming factor TGF-β1 by double antibody sandwich ELISA, both kits were purchased from Rexin Bio (Size 96T, sensitivity: <0.1 pg/mL, IL-1β test range: 1.25–40 pg/mL, TGF-β1 test range: 2.5–80 ng/mL). Operating procedure All reagents and components are first brought back to room temperature, standards, controls and samples, and it is recommended to do duplicate wells. The operating procedure is as follows:
1) Prepare the working solutions for the various components of the kit as described in the previous instructions. 2) Remove the required slides from the aluminium foil bag and put the remaining slides back into the refrigerator in a sealed self-sealing bag. 3) Set up the standard wells, 0-value wells, blank wells and sample wells. Add 50 μL of standard at different concentrations to each of the standard wells, 50 μL of sample diluent to the 0-value wells, and 50 μL of the sample to be tested to the blank wells. 4) Add 100 μL of Horseradish Peroxidase (HRP)-labelled detection antibody to the standard wells, 0-value wells and sample wells, except for the blank wells. 5) Cover the reaction plate with sealing film. Incubate the plate for 60 min at 37°C in a water bath or thermostat protected from light. 6) Remove the sealing film, discard the liquid, pat dry on blotting paper, fill each well with washing solution, leave for 20 s, shake off the washing solution, pat dry on blotting paper and repeat 5 times. If using an automatic plate washer, follow the operating procedures of the washer and add a 30 s soak procedure to improve the accuracy of the assay. At the end of the wash, before adding the substrate, pat the reaction plate dry on clean, non-flaking paper. 7) Mix substrate A and B in a 1:1 volume and add 100 μL of substrate mixture to all wells. Cover the plate with sealing film and incubate for 15 min at 37°C in a water bath or thermostat protected from light. 8) Add 50 μL of termination solution to all wells and read the Optical Density (OD) of each well on an enzyme marker.
1) Prepare the working solutions for the various components of the kit as described in the previous instructions. 2) Remove the required slides from the aluminium foil bag and put the remaining slides back into the refrigerator in a sealed self-sealing bag. 3) Set up the standard wells, 0-value wells, blank wells and sample wells. Add 50 μL of standard at different concentrations to each of the standard wells, 50 μL of sample diluent to the 0-value wells, and 50 μL of the sample to be tested to the blank wells. 4) Add 100 μL of Horseradish Peroxidase (HRP)-labelled detection antibody to the standard wells, 0-value wells and sample wells, except for the blank wells. 5) Cover the reaction plate with sealing film. Incubate the plate for 60 min at 37°C in a water bath or thermostat protected from light. 6) Remove the sealing film, discard the liquid, pat dry on blotting paper, fill each well with washing solution, leave for 20 s, shake off the washing solution, pat dry on blotting paper and repeat 5 times. If using an automatic plate washer, follow the operating procedures of the washer and add a 30 s soak procedure to improve the accuracy of the assay. At the end of the wash, before adding the substrate, pat the reaction plate dry on clean, non-flaking paper. 7) Mix substrate A and B in a 1:1 volume and add 100 μL of substrate mixture to all wells. Cover the plate with sealing film and incubate for 15 min at 37°C in a water bath or thermostat protected from light. 8) Add 50 μL of termination solution to all wells and read the Optical Density (OD) of each well on an enzyme marker.
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