Journal of neuroscience methods · 2026
Cross-species evaluation of commercial primary antibodies by Western blotting for immunohistochemical protein detection in mouse, rat, and human brain tissue.
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Medenica Tila, Bokulić Ema, Mažuranić Anton, Martinović Slavica, Judaš Miloš, Sedmak Goran
Аннотация
BACKGROUND: Commercial antibodies show variable specificity and performance, limiting reproducibility of immunohistochemical studies. We systematically evaluated antibodies used to detect brain proteins across species.
NEW METHOD: Established Western blotting (WB) and immunofluorescence (IF) validation approaches were applied systematically and in parallel to mouse, rat, and human brain tissue. The distinctive scope of the study was the comparative evaluation of 23 antibodies targeting 17 proteins, with WB results interpreted in the context of expected molecular weights, isoforms, post-translational modifications, and published evidence concerning each target. Antibody performance was further compared with publicly available validation information, and logistic regression was used to assess whether antibody citation count and manufacturer-provided validation information predicted successful experimental validation.
RESULTS: Ten antibodies (43%) showed supportive WB results, and eight (35%) were validated by both WB and IF. Several antibodies produced unexplained or non-specific bands, while others showed speciesdependent performance. Validated antibodies displayed appropriate neuroanatomical and subcellular staining patterns. Antibody citation count was moderately associated with successful validation, whereas manufacturer-provided validation information did not improve predictive performance.
COMPARISON WITH EXISTING METHODS: The overall validation rate was comparable to previously published antibody validation studies and Human Protein Atlas results. The present study extends existing work through its broader antibody panel, parallel cross-species evaluation, detailed target-protein assessment, and quantitative analysis of antibody metadata.
CONCLUSIONS: Commercial antibodies require independent validation before use in cross-species immunohistochemical studies. Our results provide a practical resource for antibody selection and highlight limitations of manufacturer claims and citation counts as indicators of antibody performance.
NEW METHOD: Established Western blotting (WB) and immunofluorescence (IF) validation approaches were applied systematically and in parallel to mouse, rat, and human brain tissue. The distinctive scope of the study was the comparative evaluation of 23 antibodies targeting 17 proteins, with WB results interpreted in the context of expected molecular weights, isoforms, post-translational modifications, and published evidence concerning each target. Antibody performance was further compared with publicly available validation information, and logistic regression was used to assess whether antibody citation count and manufacturer-provided validation information predicted successful experimental validation.
RESULTS: Ten antibodies (43%) showed supportive WB results, and eight (35%) were validated by both WB and IF. Several antibodies produced unexplained or non-specific bands, while others showed speciesdependent performance. Validated antibodies displayed appropriate neuroanatomical and subcellular staining patterns. Antibody citation count was moderately associated with successful validation, whereas manufacturer-provided validation information did not improve predictive performance.
COMPARISON WITH EXISTING METHODS: The overall validation rate was comparable to previously published antibody validation studies and Human Protein Atlas results. The present study extends existing work through its broader antibody panel, parallel cross-species evaluation, detailed target-protein assessment, and quantitative analysis of antibody metadata.
CONCLUSIONS: Commercial antibodies require independent validation before use in cross-species immunohistochemical studies. Our results provide a practical resource for antibody selection and highlight limitations of manufacturer claims and citation counts as indicators of antibody performance.