Succinate Dehydrogenase B (SDHB) Immunohistochemistry for the Evaluation of Muscle Biopsies

Abstract

Succinate dehydrogenase (SDH) is a key mitochondrial enzyme complex composed of 4 subunits. SDH histochemistry is routinely utilized in the assessment of muscle biopsies to reveal underlying pathology such as subsarcolemmal mitochondrial aggregates. In this study we evaluated the utility of SDHB immunohistochemistry (IHC) in 27 muscle biopsies, including 13 mitochondrial myopathies (MMs), 9 inflammatory myopathies (IMs), and 5 controls. SDHB IHC was performed on FFPE tissue sections with a mouse MAb (Abcam 21A11AE7) in parallel with histochemical SDH stains on fresh-frozen tissue. In all muscle biopsies SDHB IHC exhibited granular immunoreactivity and highlighted the dark type 1 and lighter type 2 staining pattern observed by histochemistry. In all cases of MM SDHB IHC showed subsarcolemmal granular aggregates involving the entire periphery of the fibers that were more distinct than those seen by SDH histochemistry. In three extraocular muscle biopsies SDHB immunoreactive speckles of various sizes were distributed throughout the entire sarcoplasm that were more prominent than those seen on SDH histochemistry. Subsarcolemmal and cytoplasmic granular aggregates seen on SDHB IHC correlated with mitochondrial pathology on electron microscopy. In cases of IM, there was diffuse sarcoplasmic SDHB immunoreactivity in degenerating fibers but no evidence of subsarcolemmal aggregates. This study demonstrates that SDHB IHC is highly sensitive and specific in the identification of MM. The automation, reproducibility, and cost efficiency of SDHB IHC offer advantages over the labor intensive histochemical method requiring frozen sections. Since this technique is performed on FFPE tissues, it can be easily applied for retrospective studies.

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“…Sections were washed, dehydrated, and cleared in xylenes. SDH staining was performed by incubating tissues at 37°C for one hour in a 0.2M sodium phosphate buffer solution [ 23 ]. Tissues were rinsed, dehydrated in graded acetone dilutions, and rinsed again.…”

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“…Briefly, sections were air-dried at RT for 10–15 min, incubated in a solution of 0.1 M phosphate buffer, pH 7.6, 0.5 mg/ml DAB (3,3′-diaminobenzidine), 1 mg/ml cytochrome c, and 2 µg/ml catalase at RT for ~30 min, washed with PBS, dehydrated, cleared and mounted under a glass coverslip (Permount; Thermo Fisher Scientific). Similarly for succinate dehydrogenase activity staining, the frozen muscle sections were air-dried for 10–15 min and incubated in SDH reaction mixture containing 1.5 mM nitroblue tetrazolium (NBT) and 50 mM di-sodium succinate in 0.2 M PBS (pH 7.6) at RT for ~20 minutes, washed with PBS, dehydrated, cleared, and mounted under a coverslip with Permount ( Punsoni et al, 2017 ). Muscle fibrosis was determined by trichrome staining (Abcam, MA).…”

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“…H&E staining was performed as previously described ( 32 ), and the percent of fibers displaying central nucleation was quantified visually on three to four cross-sectional fields of view and expressed as a percentage of the total fibers in those cross-sections. SDH staining was performed as previously described ( 53 ), and SDH activity was quantified as the gray intensity on three to four cross-sectional fields of view and expressed as percent absorbance. Additionally, SDH staining was quantified by visually classifying fibers as light, mid-tone, or dark.…”