Firstly, in order to examine the native characteristics of bovine cartilage, H and E stained tissue were visually inspected under light microscope. This was processed on newly isolated tissue in order to establish any gross changes to the histo architecture of the tissue. The articular cartilage with endothelial cell layer was also observed in this experiment. The extracellular matrix with chondrocytes showed light purple stain while cell nuclei were stained dark purple after H and E staining.
Immunohistochemical (ihc) Analysis Of The Aggrecan Molecule In Bovine Cartilage
Further characterization of the bovine cartilage tissue was carried out by immunohistochemical staining with anti aggrecan antibodies by different dilution factors. The detailed analyzed results are categorized into different sections given below:
IHC Analysis of Zinc Fixed Sections
Zinc fixed bovine cartilage tissue sections were incubated for 24 and 48 hours respectively. The immunohistochemical staining for aggrecan antibody were performed subsequently. The dilution factor of anti aggrecan antibody of 1:50 was used.
Even though the nucleus is expected to contain this protein, the dilution of antibody was low to interact with antigen in healthy cartilage and hence little staining was expected. The nuclei were stained darkly while the extracellular matrix showed light background staining all over the section uniformly.
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IHC Analysis of Zinc And NBF
IHC Analysis of NBF Fixed Sections with Various Dilution Factors.
With the exception to previous experiment conducted above, the same was continued with different dilution factors of anti aggrecan. It includes 1: 2, 1:10, 1:25 and 1:50 dilution ratios. However in this experiment, 24 and 48 hours fixed sections was carried out for immunohistochemical staining of aggrecan antibody. The extracellular matrix with nuclei showing no stain (all over the tissue) was expected and the same was proved. There was no background staining in any of the tissue sections. This included IgG1 control sections with different dilutions. The dilution ratios obtained are as follows.
The tissue with 1:2 antibody dilution factors showed best staining results. The result showed that the nuclei were darkly stained at both upper and lower surface layers. The middle layer of the tissue was lightly stained in comparison to other two layers as shown in figure 4 I and J. The antibody dilution ratios 1:10, 1:25 and 1:50 showed no stain.
IHC Analysis of NBF Fixed Sections with 48 Hours Duration
48 hours NBF fixed sections gave better results in comparison to the previous experiments. Therefore NBF fixed 48 hours sections were used. Different dilution ratios such as 1:2, 1:10, 1:25 and 1:50 was used in this experiment. The immunohistochemical staining for this NBF sections was carried out in this test.
Despite previous experiments, the results of this experiment were nearly similar. The dilution factor with 1:2 ratios was expected to have same staining as the previous experiment. However, it did not show any stain.
Due to the background staining the sections appeared to be marked positively but was not stained as predicted (as shown in figure E and F). The dilution ratios which appeared are as follows.
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