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New Insight Into the Immunostaining Method Suitable for the Pilosebaceous Complex Unit

Lee, Jaein MD*,†; Yoo, Jongman MD, PhD‡,§; Chung, Hyung Min PhD

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The American Journal of Dermatopathology: January 2016 - Volume 38 - Issue 1 - p 79-81
doi: 10.1097/DAD.0000000000000300

To the Editor:

A pilosebaceous complex unit contains both the sebaceous glands (SGs) and the hair follicle (HF) bulge regions, which are neighboring and related substructures, and it is where keratinocytes are divided into the epidermis and HF.1,2 The bulge region including HF stem cells has distinctive features; it is a permanent structure without cyclic degeneration and is protected after hair removal.3,4 Thus, the exact location of the bulge region is considered important. Moreover, agents affecting HF stem cells simultaneously affect HFs and SGs.5 And bulge stem cells and SGs are drug target and reservoirs.6 To evaluate and analyze the interactions between them, accurate immunoreactivity (IR) and differentiating the localization of the SGs and HF bulge stem cells of the overall pilosebaceous unit are also important.

However, we discovered that the amplifying signal, the advantage of the labeled streptavidin binding (LSAB) method, generally considered a more advanced immunohistochemistry (IHC) method than the avidin–biotin complex method,7 makes it paradoxically difficult for researchers to discriminate the IR patterns between the bulge cells and the transient amplifying (TA) cells in the HF. In addition, we observed that only SGs were stained in the LSAB method. Therefore, we considered presumptive causes and investigated this unintentional staining.

In this study, we investigated how the different IR patterns of the secondary antibodies in each different IHC method affect the HF substructures. Moreover, to accurately analyze the IR patterns of the HF-related substructures, we have suggested that conjugated secondary antibodies should be used instead of the LSAB method; the former have definite IR separation and no false-positive SG staining, and the latter have problems of staining only SGs that are not blocked by the avidin/biotin blocking protocol and a poor differential IR separation.

First, we confirmed the location of the SGs using Oil Red O staining (Fig. 1A). Then, using an integrin-β1 antibody as an HF stem cell marker,8,9 we investigated in detail how the IR patterns were affected, depending on the 2 different IHC methods. When using the 2-step LSAB method with the avidin/biotin blocking, an indefinite separation of IR patterns was observed; similarly, strong IR patterns were simultaneously observed in the bulge and TA cells in HF root, and moderate IR staining was observed in the SG region. The SGs showed a false-positive signal even in the negative control samples (Fig. 1B, left panel). However, when the 1-step, peroxidase-labeled conjugated antibody method was used, a more defined separation of the IR patterns of the subregions was observed without a false-positive IR in SGs; the bulge region showed the strongest IR, whereas very weak IR in the TA cells were observed (Fig. 1B, right panel). Materials and Methods are described in the Supporting Information (Supplementary Materials and Methods). The LSAB detection methods have been developed to overcome the lack of sensitivity and specificity with more intense signal amplification.7 However, the consequence, as shown in Figure 1, is that this signal amplification paradoxically leads to poor differential IR separation of the substructures of the pilosebaceous unit and that the LSAB method causes false-positive signals in SGs. In contrast, from the viewpoint of the overall pilosebaceous unit, using the 1-step conjugated antibody method results in more definite IR patterns separated by the HF-related subregions without a false-positive signal.

A, The SGs, which neighbor the bulge region, are localized by the Oil Red O staining (arrows). B, Comparison of the IR patterns on the overall pilosebaceous unit using the integrin-β1 primary antibody between (left panel) the LSAB antibody and (right panel) the peroxidase-labeled conjugated antibody. Left panel: indefinite IR separation by region with the integrin-β1 primary antibody and the LSAB secondary antibody; TA cells and the bulge cells (arrowheads) showed strong IR patterns, and the SG region showed a similarly moderate IR (arrows) because of a false-positive IR. In addition, in the negative control, the SG region showed moderate IRs (arrows). Right panel: definite IR separation by region with the integrin-β1 primary antibody and the peroxidase-labeled conjugated secondary antibody; the bulge region showed the strongest IRs (arrowheads), TA cells and SG region showed very weak and negative IRs, respectively. No IR was observed in the negative control samples. Bar, 100 μm.

We discovered that the SGs exhibited selective staining using the LSAB method even in the negative control samples (Fig. 1B, left panel). This phenomenon could result in the misinterpretation of the SGs as bulge cells when using a HF stem cell marker. Therefore, we considered the presumptive reasons and performed experiments to resolve this phenomenon with negative control samples. The avidin–biotin interaction of the LSAB method could affect the staining of certain substances in the tissue.10 We were concerned with stronger IR of the SG in the absence of the avidin/biotin blocking. Therefore, we investigated the influence of the presence or absence of the avidin/biotin blocking step; however, the SGs showed similar IR (see Figure, Supplemental Digital Content 1, We inferred that this phenomenon was regardless of avidin–biotin interaction. Also, we questioned whether SGs were sensitive to incomplete washing, one of the most common causes of background staining. The standard washing was performed 3 times for 5 minutes each, and incomplete washing was performed only once for 5 minutes with the 1-step conjugated antibody method to exclude the influence of the LSAB method itself. Interestingly, we found that the dermal stroma was the most sensitive to incomplete washing, and the stratum corneum was often weakly stained. Importantly, SG cells and keratinocytes of the epidermis and HF were relatively protected from the incomplete washing and showed little staining (see Figure, Supplemental Digital Content 1,

In conclusion, this study demonstrated that the 1-step, peroxidase-labeled conjugated secondary antibody method, and not the 2-step LSAB method, may be the IHC method of choice for pilosebaceous units. This method could be applied to acquire more discriminatory IR patterns in HF-related substructures, to prevent bulge regions from being delocalized, and to prevent false-positive SGs from being misinterpreted as bulge regions. Paradoxically, the amplifying signal as the advantage of LSAB methods, generally known as a sensitive and specific IHC method, could be disadvantageous for characterizing and localizing the bulge regions.


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