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. 2020 Jun 11;3(3):e1098.
doi: 10.1002/jsp2.1098. eCollection 2020 Sep.

Immunohistochemical analysis of protein expression in formalin fixed paraffin embedded human intervertebral disc tissues

Abbie Binch  1 , Joseph Snuggs  1 , Christine L Le Maitre  1
Affiliations

Immunohistochemical analysis of protein expression in formalin fixed paraffin embedded human intervertebral disc tissues

Abbie Binch et al. JOR Spine. .

Abstract

Immunohistochemistry (IHC) is a useful technique for the localization and semiquantification of protein expression within tissues. Adult human intervertebral disc (IVD) tissues contain a large amount of auto-fluorescence which often makes immunofluorescence techniques inappropriate on tissue samples but can be applied to isolated cell samples. Thus, IHC remains one of, if not the most common application for protein detection within IVD tissue. Immunostaining localizes antigen expression through specific epitope-antibody interactions. Within the field of IVD research, IHC is commonly used on fresh frozen and paraffin embedded tissues to elucidate the expression of antigens. Here, we discuss the principles of IHC applied to formalin fixed paraffin embedded IVD tissue and supply optimized protocols for antibodies used within our group to guide research within the IVD field.

Keywords: immunohistochemistry; intervertebral disc; protein localisation; protocol.

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Conflict of interest statement

There are no conflict of interest to declare.

Figures

FIGURE 1
FIGURE 1
Principle of Immunohistochemistry. Target antigens are detected by the application of specific primary antibodies, followed by the addition of a biotin conjugated secondary antibody which recognizes a specific sequence on the primary antibody. Localisation of bound biotinylated secondary antibody is detected by the addition of streptavidin HRP labeled ABC solution which binds to the biotin on the secondary. Enzymatic color changes occur when DAB reacts with the HRP bound to the secondary antibody, thus resulting in the detection of the target antigen
FIGURE 2
FIGURE 2
Examples of immunostained formalin fixed paraffin embedded intervertebral disc tissue sections. A, Aquaporin 7 (Abcam ab85907) cell membrane staining, canine nucleus pulposus (NP) tissue. B, Cytoplasmic Indian Hedgehog (Santa Cruz sc‐1196) staining, human NP tissue. C, Nuclear localisation of TonEBP (Abcam ab3446), human NP tissue. D, Pericellular and matrix staining of collagen type II (Abcam ab34712), human NP tissue. E, Staining of aquaporin 6 (Abcam ab191061) indicating cartilaginous endplate (CEP) localisation, canine IVD tissue. F, CD31 (Abcam ab28364) staining within human annulus fibrosus (AF) tissue indicating the presence of endothelial cell infiltration. G, CCL3 (Abcam ab32609) staining within cell clusters, human NP tissue. H, Mouse IgG3, Kappa isotype control (Abcam ab18394), human NP tissue. I, Rabbit IgG isotype control (Abcam ab37415), human NP tissue. A, B, E, F, G, H, I scale bar = 100 μm. C, D scale bar = 50 μm
See this image and copyright information in PMC

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