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Annals of Clinical & Laboratory Science 38:215-220 (2008)
© 2008 Association of Clinical Scientists

Mouse Spleen Tissue as a Staining Intensity Reference for Immunohistochemistry

Yeonsook Moon1, Gyeongsin Park2, Kyungja Han3, Chang-Suk Kang2 and Wonbae Lee4
1 Department of Laboratory Medicine, Inha University Medical College, Incheon; 2 Departments of Pathology, 3 Laboratory Medicine, and 4 Pediatrics, Catholic University Medical College, Seoul, Korea

Address correspondence to Wonbae Lee, M.D., Ph.D., Pediatrics Department, Catholic University Medical College, Holy Family Hospital, Wonmigu Sosa-dong 2, Pucheon, Kyunggi 420-717, Korea; tel 82 32 340 7045; fax 82 32 340 2255; e-mail leewb{at}catholic.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunohistochemistry (IHC) is widely used in diagnostic practice and research, but it is limited due to its subjective nature and weakness in reproducibility. For successful interpretation, IHC requires an internal reference system that controls for procedural variables and provides a staining intensity reference. We investigated the feasibility of using mouse spleen tissue as an intensity reference in conventional IHC. Formalin-fixed, paraffin-embedded mouse (BALB/c) spleen tissue was stained with variable procedural conditions including primary antibody (Ab) types, antigen retrieval methods, chromogen exposure times, and secondary Ab concentrations. Mouse spleen tissue showed identical staining intensity regardless of primary Ab types, even without primary Ab, and showed minimal differences according to retrieval methods. However, it showed various staining intensities according to chromogen exposure time and secondary Ab concentration. When mouse spleen was included in tissue microarrays and compared with the c-erbB2 IHC scoring system, splenic B cells showed weak membrane staining compatible with score 1+, whereas splenic plasma cells showed strong staining intensity compatible with score 3+. These results show that mouse spleen tissue can serve as a staining intensity reference for the interpretation of IHC.

Keywords: immunohistochemistry (IHC), mouse spleen, reference tissue for IHC, B cells, plasma cells


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immunohistochemistry (IHC) is a powerful tool that is used to identify protein expression in tissues at a micro-anatomical level; it has been used widely in diagnostic practice and in scientific research [1,2]. However, IHC has been limited by poor reproducibility of staining results and by the subjectivity of its interpretation [3,4]. There have been many discrepancies among published reports of IHC assays, which were largely caused by divergence in methodology including fixation, tissue processing, staining procedure, and interpretation [1]. Many attempts to standardize the laboratory-dependent tissue conditioning factors, including fixation and antigen retrieval, have contributed to advances in procedural and technical standardization [5–8]. As is well recognized by most pathologists, the subjectivity in interpretation gives rise to intra-/inter-observer variance, by which the significance of IHC is limited in semiquantitative and/or qualitative analysis [1,3,4,9–13]. For successful IHC staining and interpretation, an internal reference system is needed that controls for all procedural variables and that provides graded staining intensity for all primary Abs [1]. However, the development of quantifiable internal controls for IHC has proven to be very difficult [8].

Here, we used mouse spleen tissue as a new alternative for the standardization of IHC. Mouse splenic B cells and plasma cells express different amounts of mouse immunoglobulins. Therefore, formalin-fixed, paraffin-embedded mouse spleen tissue should react with the secondary antibodies (Abs), antimouse immunoglobulins, that are included in most commercial IHC kits and should show different color intensities. Theoretically, the color intensities of mouse spleen tissue could reflect IHC procedural variables, including antigen retrieval methods, chromogen visualizing times, and secondary Ab concentrations, all of which have similar effects on test tissues on the same slide. The semiquantitative results of bound primary Ab on the test tissues could be obtained by comparison with the amounts of primary Ab in the B cells and the plasma cells in the mouse splenic tissue. In the current study, we investigated the usefulness of mouse spleen tissue as an internal staining intensity reference for IHC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Paraffin-embedded blocks of mouse spleen tissue.  Four healthy BALB/c mice (8 wk old, 25 – 27 g body weight) from the Atsugi Breeding Center (Charles River Japan Inc., Tokyo, Japan) were sacrificed to obtain normal mouse spleens. The excised spleens were fixed with 10% buffered formalin for 16 hr and embedded in paraffin blocks according to a conventional tissue processing procedure.

Tissue microarray (TMA) block.  Each TMA block consisted of 40 tissue cores; 37 human breast cancers, and three mouse spleen samples. Two of the mouse spleen tissue cores were implanted in opposite corners of the block in order to monitor staining adequacy (eg, edge effect), and one was implanted in the center of the block as a reference for staining intensity.

Immunohistochemical staining.  IHC was performed on 5-µm sections of each paraffin block and TMA block. The sections were deparaffinized and rehydrated, and were then subjected to antigen retrieval performed according to several recommended methods. The endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide. The primary Ab (rabbit polyclonal anti-c-erbB2, DAKO, Glostrup, Denmark) was applied, and samples were then incubated for 90 min at room temperature. After washing, the secondary Ab was applied, and samples were further incubated for 30 min at room temperature. Finally, the slides were visualized by DAB immunostaining using the REAL EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse Kit (DAKO).

IHC intensity assay on mouse spleen tissue.  To evaluate the effects of the primary Ab, mouse spleen sections were incubated with Ab diluent only for the negative control, and with anti-leukocyte common antigen (LCA, DAKO) and anti-cytokeratin CKMNF116 (DAKO) following antigen retrieval with a pressure cooker (121°C, 3 min). The rest of the procedural conditions were identical.

Comparison of antigen retrieval methods.  Anti-cytokeratin CKMNF116 Ab was used as primary Ab. The rest of the procedural conditions were identical, except that the sections were retrieved using the following 4 methods:

  1. pressure cooker, 121°C, 3 min, in preheated sodium-citrate buffer (pH 6.0);
  2. microwave oven, 3 heatings, each 4 min, in preheated sodium-citrate buffer (pH 6.0);
  3. proteinase K (DAKO), 10 min; and
  4. pepsin (DAKO), 10 min.

Procedural variables (ie, DAB exposure time and secondary Ab concentration).  The sections were retrieved with a pressure cooker (121°C, 3 min, in pH 6.0 sodium-citrate buffer) and applied with anti-cytokeratin CKMNF116 as primary Ab. Only the visualizing step with DAKO REAL DAB+ Chromogen (Bottle C) was set differently, for 2 min, 4 min, 6 min, and 8 min. To evaluate the effects of the secondary Ab concentration, the secondary Ab, DAKO REAL EnVision/HRP, Rabbit/Mouse (Bottle A), was diluted 1:1, 1:2, and 1:4, (v/v), and was applied using identical staining conditions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Staining intensity of mouse spleen tissue according to primary Ab types.  IHC with different primary Abs on mouse spleen tissue sections showed identical staining patterns and intensities. On staining with pressure cooker antigen retrieval, the blank control staining, in which primary Ab was replaced by Ab diluent only, demonstrated appropriate staining intensity in plasma cells (Fig. 1AGo). Moreover, neither of the applied primary antibodies, anti-LCA (Fig. 1BGo) and anti-cytokeratin (Fig. 1CGo), affected the staining intensity, compared to that with Ab diluent only as a negative control. Splenic plasma cells showed strong cytoplasmic staining patterns, whereas splenic B cells showed weak membrane staining patterns on all three of the test slides.


Figure 1
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  		Fig. 1. Mouse spleen tissue showing identical staining patterns and intensities regardless of applied primary antibodies. A, antibody diluent; B, leukocyte common antigen; C, cytokeratin; each at x400 original power.

 
Staining intensity of mouse spleen tissue according to antigen retrieval methods.  We examined the staining intensities according to the conventionally-used antigen retrieval methods, including pressure cooker, microwave, proteinase K, and pepsin methods. The slides that had been treated using the four different retrieval methods showed minimal differences in staining intensity (Fig. 2Go). When the slides were treated inadequately for antigen retreival, they showed variably weak staining intensities (data not shown).


Figure 2
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  		Fig. 2. Mouse spleen, immunohistochemical staining with cytokeratin as a primary antibody, showing minimal variation in staining intensity according to antigen retrieval methods. (Panel A) pressure cooker, 121°C, 3 min, citrate (pH 6.0); (Panel B) microwave, 3x4 min, citrate (pH 6.0); C, proteinase K, 10 min; D, pepsin, 10 min; each at x200 original power.

 
Staining intensity according to IHC procedural variables.  Slides that were incubated in the DAB solution for 2 or 4 min showed weak staining intensity. However, slides incubated for 6 or 8 min showed similar and appropriately strong staining intensities (Fig. 3AGo). Slides to which 1:4-diluted or 1:2-diluted secondary Ab had been applied showed weak staining intensities, and only the slide that had been incubated with 1:1 diluted secondary Ab showed adequate staining intensity (Fig. 3BGo).


Figure 3
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  		Fig. 3. Mouse spleen tissue demonstrates variable staining intensity according to (Panel A) DAB exposure time and (Panel B) secondary antibody dilution.

 
Applications to TMA.  Mouse spleen tissue cores were integrated into a TMA block (Fig. 4AGo) to serve as controls for IHC. Specifically, two cores implanted in opposite corners of the TMA controled for the edge effect, which is frequently encountered in IHC. When stained with c-erbB2 Ab, breast cancer tissues showed variable staining intensities, case by case, with scores ranging from 0 to 3+ according to the conventional scoring system. The mouse splenic B cells showed weak membrane staining compatible with a score of 1+, whereas the plasma cells showed strong intensity compatible with a score of 3+. In the mouse spleen tissue cores, moreover, the background T cells, which were clearly negative, highlighted the weakly-stained B cells and strongly-stained plasma cells (Fig. 4BGo).


Figure 4
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  		Fig. 4. Application to TMA: (Panel A) a construct of TMA; (Panel B) staining intensity reference applied to IHC for c-erbB2 staining in human breast cancers.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of IHC are influenced by various factors, including fixation, tissue processing, antigen retrieval methods, staining conditions such as chromogen visualizing time and secondary Ab concentration, and subjectivity of interpretation [1]. There have been attempts to standardize IHC in order improve the adequacy of the IHC procedure and provide objectivity in interpretation [13,14]. However, an internal staining intensity reference on the same slide as the test tissue, to reflect the tissue processing and staining conditions of the test tissue, has not been available. The rationale of this study is based on the fact that there are numerous B cells and plasma cells in the mouse spleen, and mouse immunoglobulin is expressed on these B cells and in the cytoplasm of plasma cells. Most commercially-available IHC detection kits provide secondary antibodies that contain anti-mouse immunoglobulin. Therefore, mouse spleen tissue could be useful as a reference tissue showing two staining intensities, one for the B cell membrane and another for strongly positive staining in the plasma cell cytoplasm. In this study, mouse spleen tissue showed constant staining intensity regardless of applied primary Abs, even in the absence of primary Ab. In addition, mouse spleen tissue showed a similar staining pattern and intensity with minimal variance according to the four antigen retrieval methods evaluated. In this study, mouse spleen tissue located on the same slide as test tissue showed the effects of such variables. These results provide the basic rationale for the utilization of mouse spleen tissue.

Monitoring the IHC procedural variables is one of the most important requirements for quantitative IHC [1]. One approach that has been suggested is the Quicgel method, in which cultured cell lines expressing known and independently measured quantities of target molecules are simultaneously formalin-fixed and paraffin-embedded alongside an experimental specimen [11]. This method might be extremely useful in a prospective study for a readily-designed target molecule, but is obviously limited in terms of general use for various primary Abs [13]. In this study, mouse spleen tissue controlled for the procedural variables, including the adequacies of antigen retrieval, DAB exposure time, and secondary Ab concentration, which satisfies one of the important requirements for an internal reference used in the interpretation of quantitative IHC.

In the interpretation of IHC for some target molecules, including c-erbB2 and EGFR, intensity grading is critical [15,16]. In most investigations that have utilized IHC, a scoring system has been used for data analysis [10]. The interpretation variance is a longstanding issue in IHC. A two or more blind-observer system is an easy and simple method by which to yield final results by mutual agreement. Although this method provides some advantages in terms of objectivity of data, it does not eliminate the basic problems of subjectivity and intra-/inter-observer reproducibility [3,4], and also does not fully reflect the procedural variables. Computer-aided image analysis systems provide outstanding accuracy and reproducibility in the quantitative IHC assay [9,12]. They faithfully a sure objective interpretation of the state of the staining itself, if not for procedural variables influenced by different staining times or in different laboratories. However, the determination of the cut-off value for positivity and/or the scoring system remains subjective by nature.

In this study, when stained with c-erbB2 Ab and compared to the c-erbB2 IHC scoring system in breast cancer, mouse splenic B cells showed weak membrane staining compatible with score 1+, while the plasma cells showed strong staining intensity compatible with score 3+. These results show the potential role of mouse spleen tissue as a graded staining intensity reference.

TMA is a formidable tool for high-throughput study using IHC. When TMA is applied, mouse spleen tissue works as a reporter core for the overall staining process, including the edge effect, and also acts as a reference sample that makes it possible to compare and integrate the results of IHC staining at different times and in different laboratories.

In conclusion, we have demonstrated that mouse spleen tissue shows constant staining intensity regardless of the applied primary Ab, and shows minimal variance in staining intensity according to retrieval methods. Moreover, it reflects IHC procedural variables, including adequacies of antigen retrieval, chromogen exposure time, and secondary Ab concentration. The mouse spleen tissue shows two constant staining intensities: membrane staining of B cells, and strong cytoplasmic staining pattern of plasma cells in every slide. These results show the usefulness of mouse spleen tissue as an internal staining intensity reference for IHC.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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