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A novel immunohistochemistry slide adhesive 3-(trimethoxysilyl) propyl methacrylate (tpm) for bluetongue virus serotype-1 antigen detection in ovine foetal tissues Babu Prasath N.1, Singh Karam Pal1*, Saminathan M.1, Sahoo Monalisa1, Biwas Sachay Kumar2, Kumar Asok M.1, Patel Mahendra Sagar1, Singh Rohit1 1Division of Pathology, ICAR-Indian Veterinary Research Institute, Bareilly, Izatnagar, Uttar Pradesh 2Eastern Regional Station of ICAR-Indian Veterinary Research Institute, Kolkata, India. Address for Correspondence Dr Karam Pal Singh, Centre for animal disease research and diagnosis (CADRAD), ICAR-Indian Veterinary Research Institute, Bareilly, Izatnagar-243122, Uttar Pradesh, India, E-mail: karamcadrad62@gmail.com
Abstract The present study describes the use of 3-(trimethoxysilyl) propyl methacrylate (TPM) as a glass slide adhesive for better tissue adherence in immunohistochemistry. TPM is an alkoxysilane, dissolved in acidified water (pH 3.5) with glacial acetic acid. TPM formed a gel cast on the glass slide surface, which enhanced the net positive charge of the slide. Tissues with net negative charge adhered with gel cast adhesive on the slide surface with covalent bonding. Practically, foetal tissues have less tissue adherence capacity due to less net negative charge. Foetal brain, pancreas, lymph node and umbilical cord of sheep were included in the study. Foetal tissues were collected and fixed in Bouin’s fixative and processed as per standard paraffin embedding technique. Immunohistochemistry was performed on foetal tissues and umbilical cord using three different glass slide adhesive such as poly-L-lysin (PLL), aminopropyl triethoxy silane (APES) and TPM under different antigen retrieval system. TPM significantly enhanced the foetal tissue adherence, when compared with commercially available adhesives such as PLL and APES. In the present study, foetal tissues adhered with TPM coating well tolerated the harsh proteinase K antigen retrieval treatment. Comparatively, TPM coated slides revealed mild to zero tissue loss and less to no background staining. In addition, TPM was cost effective as many commercially available slide adhesives were either costlier or needed accessory chemicals for coating which in turn added the cost per slide. The present study showed that TPM was cost-effective, less hazardous, less time consuming, easy-to-use adhesive for manual preparation of coating slides when compared to other protein adhesive systems. The present experiment also emphasized that tissues with less adherence capacity could be coated with TPM adhesive for best IHC results. Top Keywords Histology, Immunohistochemistry, Slide adhesive, 3-(trimethoxysilyl) propyl methacrylate. Top | Introduction Immunohistochemistry (IHC) is a powerful methodology used more frequently in veterinary laboratories for both diagnostic and research purposes. Though IHC is complex, but it is a more sensitive method for detection of antigen in tissues. IHC involves both immunologic and chemical reactions for detection of antigen using primary and secondary antibodies and finally labelling of immunoglobulin with substrate and enzyme to produce coloured complex, respectively1. Tissues are subjected to various physical and chemical influences to visualize the antigen under microscope. A critical requirement is to keep the tissues intact on the glass slides from initial deparaffinization up to the antigen visualization. Tissue adherence on slide surface play a principal role in maintaining tissues on slides during IHC processing. For over the years, various slide adhesives have been used to increase the tissue adherence on glass slides and to prevent separation in whole or any of the IHC processing steps2. Commercial adhesives are used in most laboratories and amongst the most frequently used is poly-L-lysine (PLL)3 and aminosilane (3-aminopropyltrioxysilane) (APES)4. Various factors including the cost determine the usage of these adhesives for IHC. In the present communication, we describe the usage of 3-(trimethoxy silyl) propyl methacrylate (TPM) as an effective tissue adhesive, especially for foetal tissues fixed in Bouin’s fixative to detect blue tongue virus antigen. The study compared the efficiency of APES and PLL coated slides with manually prepared TPM coated slides with regard to cost effectiveness, tissue adherence capacity, different antigen retrieval methods and background staining. |
Top Materials and Methods Preparation of microscopic glass slides for coating Microscopic glass slides (Bluestar©) were placed in a plastic tray and washed twice with tap water and once with distilled water to displace visible dirt on slide. Later, slides were soaked into a detergent water (Surf excel liquid, 2 ml per litre of water) for 30 minutes (min) to remove dirt and grease over glass slide surface. Further, glass slides were washed with tap water twice to remove excess detergents. Slides were transferred and racked into slide rack and washed with running tap water for 2 hours (h) until foam stopped coming out and the slides were left in water overnight to dissolve and remove remaining detergents over glass slides. Later, slides were rinsed with distilled water twice and later drained. Coating with TPM Slide coating solution was prepared by dissolving 4 ml of TPM in distilled water having pH 3.5 (pH was adjusted with glacial acetic acid). Added TPM was mixed vigorously until solution became clear in acidic solution. Freshly prepared TPM coating solution was poured over the slides in slide rack and the slides were allowed to immerse completely in TPM coating solution. After 1 h, the coating solution was poured oft and the glass slides were rinsed with distilled water to remove excess TPM on slides. The slides were dried completely in the incubator at 37°C for 1 h. After complete drying, the slides were stored at 4°C until further use. Fixation of foetal tissue Foetal tissues were fixed in Bouin’s fixative using buffered saline for 12 hours. Later, picric acid from the tissues was removed by washing in 70% ethanol followed by tap water overnight. Tissues were also preserved in routine 10% neutral buffered formalin for comparison. Fixed tissues were processed as per standard paraffin embedding technique5. IHC protocol IHC procedure was followed as per the standard protocol using non-avidin-biotin system6. Briefly, deparaffinization was carried out in xylene, three changes of 10 min each followed by hydration with descending grades of ethanol for 5 min each and one washing in distilled water. Later tissue sections were allowed for sequential washing as follows, 1) Once in 1xPBS, 2) thrice in 1xPBS-T (phosphate buffered saline with Tween-20) and 3) once in 1xPBS, each change for 5 min. Antigen retrieval was carried out by both physical and enzymatic methods. Proteinase K (10 mg/mL; 6 pl/mL dilution) was used and incubated at 37°C for 35 min. Heating tissue sections in micro-oven at 150°C for 3–5 min intermittently, with antigen unmasking solution (Vector laboratories) was used as a physical method. Antigen retrieval was followed by sequential washing as mentioned above. Then, the tissue sections were quenched with 3% hydrogen peroxide in ethanol at room temperature for 1 h in a dark chamber followed by sequential washing. Endogenous blocking was done with horse serum (1:10 dilutions) (Sigma Alrich®) at 37°C for 1 h followed by washing with 1xPBS twice for 5 min each. Later, primary antibody (rabbit hyperimmune sera raised against BTV-1 core antigen) was added sufficiently to cover entire tissue section at 1:10 dilution and incubated at 4°C overnight. Ater incubation, the tissue sections were subjected to sequential washing as previously mentioned. Negative control was kept for each section on the same slide by incubating with 1xPBS instead of the primary antibody. HRPO conjugated goat anti-rabbit secondary antibody (Sigma Aldrich®) was added to both the sections (1:200 dilution) and incubated at room temperature for 45 mins followed by sequential washing as mentioned previously. Working chromogen (diaminobenzidine, DAB, TaKaRa) was added and incubated for 1 min. Chromogen reaction was stopped by placing tissue sections in distilled water. Tissue sections were counter-stained with Mayer’s haematoxylin (Abcam®) for 2–5 min and sections were mounted with aqueous mount. Tissue sections were then visualized under microscope for specific positive signal. Tissue selection and grading More delicate tissues which fail to adhere firmly with commonly used adhesive such as foetal brain, pancreas, lymph node and umbilical cord of sheep were also included in the present study. 4x3x2 (number of tissues x number of adhesives x number of antigen retrieval methods) sets of tissue sections were used to compare effect of PLL, APES and TMP on tissue adherence with both physical and chemical antigen retrieval methods. The ability of the glass slide to tightly adhere the tissue section was evaluated on a scale of 1 to 47 as 1-low adhesion (tissue loss 50% and above), 2- medium adhesion (tissue loss 10–50%), 3-Moderate adhesion (tissue loss 10% and less) and 4-strong adhesion (no gross tissue loss). The effect of different antigen retrieval methods on BTV-1 using three types of adhesives was scored based on the colour intensity of positive signals in tissues from + to ++++ as (+) mild, (++) medium, (+++) moderate and (++++) strong intensities. The back ground staining of different tissue adhesives was scored from 0 to 3 as 0- no back ground staining, 1- mild, 2- medium and 3- strong back ground staining. The cost effectiveness between three different adhesives was calculated based on their commercial price, accessary chemicals used while coating the glass slides and the number of slides coated. Top Results Regarding adherence, tissue samples on PLL coated slides showed mild to moderate loss in heat induced antigen retrieval and mild but focal tissue loss in proteinase K treated tissues. On the other hand, tissues in APES coated slides showed moderate loss in heat induced antigen retrieval whereas tissue loss was mild in enzymatic treatment. In total, less than 10% tissue loss was evident in both PLL and APES coated slides. Parallelly, tissue sections on TMP coated slides revealed strong adherence without gross loss of tissues except mild focal loss of brain tissue which was noticed in heat induced antigen retrieval. Comparatively, tissues showed moderate to strong adherence capacity towards enzymatic digestion with proteinase K in all the coated slides which has been summarized in Table 1. |
Comparing the antigen retrieval system by enzymatic and physical method, proteinase K retrieved BTV-1 epitopes more efficiently than heat induction in foetal tissues. Though there was no comparative difference noted between slide adhesives, colour intensity of positive antigen signal was intense in PLL and TMP coated slides. The heavy and harsh method of proteinase K digestion showed excellent antigenic epitope retrieval in foetal tissues fixed in Bouin’s fixative than physical method which has been shown in Table 2. |
Non-specific back ground staining was scored with different antigen retrieval system and compared between three glass slide adhesives. Mild to no back ground staining was noticed in PLL and TMB coated slides (Figure 1–4). Non-specific signalling was more evident in APES coated slides as compared to PLL and TMB which has been summarized in Table 3. Among the tissues, umbilical cord showed intense non-specific staining in all the coated slides. In general, non-specific signalling was comparatively less pronounced in proteinase K treatment than heat treatment. |
Price of the glass adhesive is a major constraint. Based on the commercial prices of the available chemicals, TMP was comparatively cheaper. Additionally, preparation of APES coated slides requires accessary chemicals such as acetone for dilution, whereas TMP was diluted in water which reduced additional cost. The quantity required for coating 200 slides was comparatively higher in PLL followed by APES and least in TMP. 4 ml TMP per litre of water was sufficient for 200 coatings. |
Top Discussion Staining procedure is a major counterpart in pathology laboratories. It includes routine haematoxylin and eosin staining, special staining procedures to recognise unique features in tissues and immunohistochemistry. IHC play a crucial role in diagnosis of infectious disease and cancers in terms of specificity1,8. |
IHC is a quite complex process where tissues are subjected to highly variable temperatures, prolonged incubation with various immunological and chemical reactions. Further, long exposure to solvents during deparaffinization, aqueous solutions inclusive of several vigorous washing with water and detergent solutions (Tween 20, Triton X 100, saponin), oxidative reaction of tissue with hydrogen peroxide and harsh treatment such as boiling at high temperature or tissue digestion with enzymes (protease K, trypsin, pepsin, pronase E or ficin)9‘10‘11 are included. Affinity of tissue to withstand such extreme changes is wholly dependent on the type of slide adhesive used to coat the slides and the properties of tissue adherence12,13. |
Traditionally, egg albumin in glycerine and gelatin are in use for routine histological procedures9. Lately, various commercial glass adhesives are available namely poly-L-lysin, aminosilane (APES), protected isocyanate and various cationic polymers2,3,4. Amongst these, PLL and APES are the most commonly employed in many pathological laboratories. All of these chemicals are used to coat slides in order to address tissue adhesion problem specially when sections are incubated with proteases to unmask over-fixed antigens. |
Tissues get adhered to slide surface due to its net negative charge and are attracted to positive charge created on glass slide by either ionic or covalent interaction between tissue and adhesive. Slides with positive charge created by adhesives are known as plus (+) slides12. In practise, most clinical laboratories use commercially available coated slides. |
These positively charged glass slides show convincing results for the majority of tissues but not for all types. Net negative charge of tissue depends on the number of peptides or proteins it harbours. Tissue with less or small peptide or protein may have too litle net negative charge and so are poorly adhesive to conventional glass adhesives. Highly delicate tissue with less tissue adherence on conventional coatings includes bone marrow specimens, fatty tissues and brain2. In practise, a few tissues notably the spinal cord, pancreas and foetal tissues are highly notorious for their reluctance to adhere firmly to the glass surface of commercially available coated slides3. Hence, we experimented IHC for detection of bluetongue virus antigen in foetal tissue from sheep experimentally infected with bluetongue virus serotype 1 during pregnancy. |
3-(trimethoxysilyl) propyl methacrylate (TPM) is an alkoxysilane with molecular formula C10H20O5Si having molecular weight 248.35. It is a colourless transparent liquid and is soluble in a variety of solvents such as methanol, ethanol, isopropanol, acetone, benzene, toluene and xylene. Obviously, it reacts with water (pH ~ 4.0) and remains soluble after hydrolysis14. |
It is widely used as a silane coupling agent for unsaturated polyester-fiberglass composites15, copolymerized with styrene in formation of sol-gel composites16, in microparticle surface modification and employed in dental polymer composites16. In addition, it is also used in adhesives to increase bonding properties. It works the best when surfaces are typically treated with 0.5% (v/v) concentration in ethanol or acidic water with pH 3.5. |
TPM seems outdated and is rarely used in clinical laboratories for IHC. Moreover, due to the commercial availability of PLL and APES coated slides, its use is limited. Experimentation in the present study revealed that the effectiveness of tissue adherence was comparatively greater with TPM as compared to other protein adhesive systems. As per the literature, upon adsorption, it forms a monolayer on the glass surface by hydrogen bonding through the carbonyl group of the silane and later, silane multilayers are formed with predominant free carbonyl groups which covalently link to form polyacrylamide gels on glass surface. The gel cast formed over glass slide do not lift from the glass surface as a result of shrinkage or swelling due to pH gradient formation during isoelectric focusing which could be a reason for the superiority in adhesive property than other adhesives18. |
TPM aids in easy positioning of tissue sections on glass slide surface while retrieving from water bath due to its less immediate tissue adherence as evinced by Dyanov and Dzitoeva19. In the present study, the TPM was found to be advantageous as there was minimal to nil gross tissue loss from the slides while performing IHC. It was noted that tissue adherence by TPM was long-lasting and the coated slides with tissues were practically effective when stored covered at room temperature for more than 10 days. The coated slides remained useful for longer period when stored at 4°C. The added advantage of TPM coating was that water with acidic pH (pH 3.5) was sufficient to coat the slides satisfactorily when compared to acetone diluted solution in APES. PLL on the other hand was ready to coat instantaneously without dilution, but higher cost was the constraint. Collectively, coating with TPM could be done with minimal time and cost when compared to other protein adhesive system. All tissues reported to have less adherent capacity showed minimal to zero tissue loss in the procedure followed with TPM. It was clearly evident that TPM formed gels on slide which remained attached during harsh, adverse processing protocols and staining procedures. Though background staining was mild to nil in all the tissues, the umbilical cord showed high background staining. Diffuse extracellular matrix with high collagen, hyaluronic acid and glycoprotein of umbilical cord might be responsible for the enhanced background staining20. |
The cost of production was relatively less with TPM due to its lower commercial price than PLL and APES. Amongst all, TPM was less hazardous causing mild irritation during inadvertent usage. Enzymatic digestion for antigen retrieval was the harshest reaction in IHC of tissues for complex antigen epitopes especially when tissues were preserved with strong fixative and for prolonged duration. Enzymatic tissue digestion was practically followed for such tissues. In the present study, the effectiveness of TPM was greater with picric acid fixed tissues and proteinase K antigen retrieval system showed nil gross tissue loss. |
Top Conclusion We conclude that use of TPM showed effective tissue adherence to foetal tissues with minimal to zero gross loss. It is advantageous over other protein systems due to lesser cost, easier manual preparation of slides, minimal loss of tissues by enzymatic digestion and lesser background staining with intense positive signals. Therefore, we recommend the use of TPM coated slides for IHC for all type of tissues for best outcome. |
Top Acknowledgement We thank the Director, Joint Directors and Head, Division of Pathology, ICAR-Indian Veterinary Research Institute for providing all the facilities to carry out this research work. |
Top Figures | Fig. 1.: Positive immunolabelling of BTV-1 in capillary cells of cerebrum of foetal brain in TPM coated slide ×200;
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| | Fig. 2.: Positive immunoglobulin of BTV-1 in acinar cells of exocrine gland of foetal pancreas in TPM coated slide ×400;
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| | Fig. 3.: Positive immunoglobulin of BTV-1 in histiocytes of mesenteric lymph nodes in TPM coated slide ×200;
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| | Fig. 4.: Positive immunoglobulin of BTV-1 in cord erythrocytes with in umbilical cord in TPM coated slide ×200.
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Tables Table 1.:
| | Antigen retrieval method | PLL Brain | Pancreas | Lymph node | Umbilical cord | APES Brain | Pancreas | Lymph node | Umbilical cord | TMP Brain | Pancreas | Lymph node | Umbilical cord | | Proteinase K | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 4 | 4 | 4 | 4 | | Heating | 3 | 3 | 3 | 3 | 2 | 2 | 3 | 3 | 3 | 4 | 4 | 4 |
| | Table 2.:
| | Antigen retrieval method | PLL Brain | Pancreas | Lymph node | Umbilical cord | APES Brain | Pancreas | Lymph node | Umbilical cord | TMP Brain | Pancreas | Lymph node | Umbilical cord | | Proteinase K | ++ | +++ | +++ | ++ | ++ | ++ | +++ | ++ | ++ | ++++ | ++++ | ++++ | | Heating | + | ++ | ++ | + | + | ++ | ++ | + | + | ++ | ++ | ++ |
| | Table 3.:
| | Antigen retrieval method | PLL Brain | Pancreas | Lymph node | Umbilical cord | APES Brain | Pancreas | Lymph node | Umbilical cord | TMP Brain | Pancreas | Lymph node | Umbilical cord | | Proteinase K | 1 | 0 | 0 | 2 | 0 | 1 | 1 | 1 | 0 | 0 | 0 | 1 | | Heating | 1 | 0 | 0 | 2 | 1 | 2 | 2 | 2 | 1 | 0 | 0 | 2 |
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