Abstract

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Aim: to optimize Tenascin-C immunohistochemistry staining protocol for rat cardiac tissue
Methods: This is a preliminary experimental study at Histology Lab Faculty of Medicine University of Indonesia from 7th December 2007 to 2nd July 2008. Samples were paraffin sections of rat cardiac tissues, and paraffin sections of brain tissue were used as positive control and absence of overnight primary antibody incubation as negative control. Formalin fixation was used before paraffin blocks were made. Immunohisto-chemistry protocol was modified from Hannekamp, et al, who stained human tissue.Staining results were recorded and analyzed. Staining was considered successful when the positive and negative controls were positive and negative respectively. If the protocol was considered unsuccessful, the data obtained and the steps in the protocol was reviewed and analyzed for cause/s of failure and troubleshooting steps were taken.
Results: The staining results according to the protocol and subsequent troubleshooting protocols showed mostly false positive results, which cause was supposed to be the cross reaction of secondary antibody with the rat tissue.
Conclusion: In starting a new immunohistochemistry staining for paraffin sections, care should be paid on the compatibility of the kit for paraffin section, choice of primary antibody that react with the antigen to be studied, and choice of secondary antibody that do not react with the tissue to be studied.

Introduction

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Immunohistochemistry technique is the visualization of a tissue or cellular component in situ by detecting specific antigen using antibody-antigen interactions where the antibody has been tagged with a visible marker. The marker may be a fluorescent dye, colloidal metal, hapten, radioactive marker or an enzyme that digest a substrate to reveal the substrate color.
Optimal visualization of a certain protein occurs when higher signal to non-specific background (noise) ratio is achieved. Therefore, signal amplification and reduction of non-specific background staining is the ultimate strategy to obtain optimal result.
Signal amplification may be performed using labeled StreptAvidin-Biotin (LSAB). In this method, there are three reactions. The first reaction is between a non-labeled primary antibody and specific antigen in tissues/cells. The second is between the primary antibody and a biotinylated secondary antibody, and the final reaction is between biotin and horse radish peroxidase-conjugated Streptavidin (HRP-StreptAvidin). The peroxidase will then oxidize a chromogen substrate that will appear as a brownish substance.Tenascin-C (TNC) is an extracellular matrix glycoprotein that may be found in stem cell microenvironment (stem cell niche) at bone marrow and subventricular zone of the brain and possibly at other organ as well [1, 2]. Tenascin-C was first isolated from embrionic tissue, while in adult tissue TNC expression remains at epithelial-mesenchymal junction. Tenascin C is known as regulator for several post-natal cellular functions, e.g: cell growth, T cell suppression, and wound healing [3]. Other than that, TNC also play a role in hemmaglutination, inflammation and progression towards invasive cancer. These findings harbored the significance of tenascin C expression at tissue hotspots, making it an interesting protein to investigate. In order to begin studies on tenascin C expression, we first aimed to optimize Tenascin-C immunohistochemistry staining protocol for rat cardiac tissue.
Methods
This is a preliminary experimental study to optimize TNC immunohistochemistry staining, performed at Histology Lab Faculty of Medicine University of Indonesia from 7th December 2007 to 2nd July 2008. Samples were paraffin sections of rat cardiac tissues, and paraffin sections of brain tissue were used as positive control and absence of overnight primary antibody incubation as negative control. Formalin fixation was used before paraffin blocks were made. Protocol of the whole study was approved by Ethical Commission for animal studies Ministry of Health Republic of Indonesia number LB.03.02/KE/1529/2008. Immunohistochemistry protocol was modified from Hannekamp, et al [4].Staining results were recorded and analyzed. Staining was considered successful when the positive and negative controls were positive and negative respectively. If the protocol was considered unsuccessful, the data obtained and the steps in the protocol was reviewed and analyzed for cause/s of failure and troubleshooting steps were taken.
Immunohistochemistry staining protocol
Protocol for immunohistochemistry staining procedure included de-paraffinization, rehydration, blocking of endogenous peroxidase activity using hydrogen peroxide, antigen retrieval, blocking of non-specific background, treatment with primary antibody, secondary antibody, visualization of antigen-antibody reaction, counterstaining and mounting.
De-paraffinization and re-hydration
The parafin sections were placed on to glass slide, and deparaffinisation was done by immersing the glass slide in xylene 2 times for 5 minutes each. Subsequent rehydration in graded alcohol concentration was done using absolute alcohol, alcohol 95% and alcohol 80%; each for 5 minutes.
Blocking of endogenous peroxidase activity
Blocking of endogenous peroxidase activity was done inside a moist chamber. Every slide was covered with 4% v/v H2O2 in methanol for 20 minutes, followed by washing in running tap water for 3 minutes
Antigen retrieval
The slides were placed inside a Coplin jar that was filled with citrate buffer pH 6.0 and heated in a microwave until reaching boiling point for 5 minutes, and the heating was repeated three times. Then the slides were let to cool in room temperature for 45 minutes, and wash by repeated dipping in PBS pH 7.4 three times for 3 minutes each.
Blocking of non-specific background
Blocking serum: blocking of non-specific background staining was done using normal rabbit serum (10% v/v serum in PBS pH 7.4 for 20 minutes
Treatment with primary antibody and secondary antibody
Dilution of primary antibody: anti-human TNC (N19) goat IgG (Santa Cruz sc9871), 1:200 was done in PBS pH 7.4 mixed with 1% v/v blocking serum. For treatments with antibodies, the slides were placed in a moist chamber. Primary antibody treatment was done by incubating the slides with diluted primary antibody at 40 C overnight. The next day, the slides were washed in PBS pH 7.4 twice for 3 minutes each. Incubation with secondary antibody (biotinylated anti-goat swine IgG, LSAB kit, Dako k-0679) was done at room temperature for 15-30 minutes. After that, excess solution was wiped off from the slides.
Visualization of antigen-antibody reaction
Visualization of antigen-antibody reaction was done by incubation with streptavidin-HRP for 15-30 minutes, then the slides were washed with PBS pH 7.4 twice for 3 minutes each, and excess solution was wiped off from slides. Further, incubation with DAB chromogen substrate was done at room temperature for 10 minutes, followed by washing with PBS pH 7.4, and then with running tap water.
Counterstaining and mounting
Counterstaining was done with Lily Mayer’s Hematoxylin for 2 minutes, followed by step wise dehydration using graded alcohol concentration: 95% and absolute alcohol twice, each for 5 minutes. After that, clearing was done twice with Xylene for 5minutes each, and finally the slides were mounted with Entelan.
Analysis of data
When false positive results were obtained: the procedure was reviewed for possible causes, i.e. antibodies were too concentrated and washing was not optimal, peroxide treatment was not optimal either in the concentration of the peroxide, time of incubation, or the peroxide had lost its activity, or blocking of non specific background was not optimal.
When false negative results were obtained: the procedure was reviewed for possible causes, i.e. de-paraffinization and re-hydration was not optimal, antigen retrieval was not optimal, dilution of primary antibody was over diluted, or incubation time of primary antibody was too short.
After the protocol was reviewed, and cause of failure supposed, subsequent modifications of protocol were made. Staining results from the protocol and subsequent modifications were recorded and compared to obtain the optimized protocol.
Results
The staining results according to the protocol showed positive staining on all of the slides, including the negative control (false positive). Troubleshooting to develop subsequent procedure was done, and overall, there were 4 modifications of the protocol (Table 1).
Result of modification 1 was false negative. However, staining of cardiac tissue showed inconsistent staining, and some showed positive staining on cytoplasm and nucleus of cardiomyocytes (intracellular components).
Further, modification 2-4 mostly showed inconsistent results with mainly false positive results. As mostly of the staining gave false positive results, we checked the specification of secondary antibody, and we found out that the secondary antibody in LSAB kit Dako-0679 (anti-goat swine IgG) is a non- specific antibody as it can cross react with rabbit and mouse IgG.
Discussion
In this study, the primary antibody was goat IgG against human TNC, which cross react to mouse and rat TNC according to the manufacturer. Though the primary antibody is not recommended for immunohistochemistry staining, it was used by Hanekamp et al for immunohistochemistry staining of human tissue with good result [5].
When the protocol according to modified protocol of Hannekamp et al yielded false positive result, we supposed that the in-optimal blocking of endogenous peroxidase activity and nonspecific background was the cause. Therefore, in modification-1 protocol, we change the peroxidase with that from the kit, and the blocking serum with normal horse serum. In addition, we use de-ionized instead of tap water for the washing step, and we got false negative results. However, some of the cardiac tissue showed positive intracellular staining. At the time of experiment, we supposed that the positive staining was nonspecific background (noise), as a previous study showed tenascin-C as extracellular/ intercellular molecules [5], though in our later finding using Santa Cruz staining kit that gave good result and consistent Tenascin C staining in rat tissue, we found tenascin C expression in cardiomyocytes [6]. We supposed the false negative result was due to improper de-paraffinization and re-hydration that caused in-optimal antigen retrieval and failure in antigen-antibody reaction. In addition, nonspecific background may be due to high concentration of primary antibody. Therefore, in modification-2 protocol we elaborate the de-paraffinization and dehydration steps, but we reduced the amount of primary antibody, and indeed we got positive results, but also on the negative controls.We supposed that the false positive result was due to prolonged exposure to the primary antibody, which caused insufficient washing, and trace primary antibody was still found on the specimen. Therefore, in modification-3 and -4protocol we used the first concentration of primary antibody and we either reduced the time to 30 minutes or overnight, but and we still got mostly false positive results. As the most of our result showed false positive result, we supposed that the cause was the secondary antibody that we did not suspect before. Therefore, in starting a new immunohistochemistry staining, not only the primary antibody and the compatibility of the kit for paraffin section should be taken into account, but also the nature of secondary antibody. In this preliminary study, we have been not careful in choosing the kit, and not careful in reading the property of the kit. Our careless made us used a kit that contained a secondary antibody that according to the manufacturer cross reacts with mouse tissue, while the animal we used are rat; so the secondary antibody may react with rat tissue as well, thus the mostly false positive results.
conclusion
In starting a new immunohistochemistry staining for paraffin sections, care should be paid on the compatibility of the kit for paraffin section, choice of primary antibody that react with the antigen to be studied, and choice of secondary antibody that do not react with the tissue to be studied.

References

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Source(s) of Funding

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The funding of this study was supported by the university of Indonesia Master’s degree Research Grant, contract number: 243C/DRPM-UI/N1.4/2008

Competing Interests

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Competing interest none declared

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