Secondary reagents for immunohistochemical research of rat brain

Abstract

BACKGROUND: The critical factor when working with immunohistochemistry in laboratory animals is to select appropriate secondary antibodies, that allow clear and specific visualization of tissue antigens. Many reliable secondary reagents are currently not available for purchase, which determines the high relevance of replacing them with other detection systems.

AIM: To verify the effectiveness of available secondary reagents for immunohistochemical research of the rat brain.

MATERIALS AND METHODS: Brain samples from Wistar (n=2) and SHR (n=2) were used for the study. Iba-1, GFAP and vimentin immunohistochemistry was carried out using various polymer-based detection systems, namely UltraVision Quanto Detection System HR, N-Histofine Simple Stain MAX PO and UnoVue Rabbit HRP.

RESULTS: All three studied polymer systems demonstrated visualisation of target proteins in brain tissues and cells corresponding to the general understanding of structures containing Iba-1, GFAP and vimentin. The UnoVue Rabbit HRP and UltraVision Quanto Detection System HRP kits showed good and similarly specific immunohistochemical reaction. However, the UnoVue Rabbit HRP kit was less sensitive compared to the UltraVision Quanto Detection System HRP. The reaction with N-Histofine Simple Stain MAX PO was not optimal due to the presence of non-specific background staining that was not present with other reagents. Apparently, this is due to the focus of the kit on immunohistochemical staining of human tissue and the likely absence of an antibody purification with rat serum.

CONCLUSIONS: Two secondary antibody kits from the three studied showed optimal efficiency of immunohistochemical reaction and minimal background staining. N-Histofine Simple Stain MAX PO is not suitable for immunohistochemical research of rat brain tissue.

Keywords

antibody specificity / immunohistochemistry / laboratory rats

References

[1] Bordeaux J, Welsh A, Agarwal S, et al. Antibody validation. Biotechniques. 2010;48(3):197–209. Corrected and republished from: Biotechniques. 2010;48(5):351. doi: 10.2144/000113382

[2] Bordeaux J., Welsh A., Agarwal S., et al. Antibody validation // Biotechniques. 2010. Vol. 48, N 3. P. 197–209. Corrected and republished from: Biotechniques. 2010. Vol. 48. P. 351. doi: 10.2144/000113382

[3] Gown AM. Diagnostic immunohistochemistry: what can go wrong and how to prevent it. Arch Pathol Lab Med. 2016;140(9):893–898. doi: 10.5858/arpa.2016-0119-RA

[4] Gown A.M. Diagnostic immunohistochemistry: what can go wrong and how to prevent it // Arch Pathol Lab Med. 2016. Vol. 140, N 9. P. 893–898. doi: 10.5858/arpa.2016-0119-RA

[5] Mao S, Xiong G, Johnson BN, et al. Blocking cross-species secondary binding when performing double immunostaining with mouse and rat primary antibodies. Front Neurosci. 2021;15:579859. doi: 10.3389/fnins.2021.579859

[6] Mao S., Xiong G., Johnson B.N., et al. Blocking cross-species secondary binding when performing double immunostaining with mouse and rat primary antibodies // Front Neurosci. 2021. Vol. 15. P. 579859. doi: 10.3389/fnins.2021.579859

[7] Keifer J, Summers CH. Putting the “biology” back into “neurobiology”: the strength of diversity in animal model systems for neuroscience research. Front Syst Neurosci. 2016;10:69. doi: 10.3389/fnsys.2016.00069

[8] Keifer J., Summers C.H. Putting the “biology” back into “neurobiology”: the strength of diversity in animal model systems for neuroscience research // Front Syst Neurosci. 2016. Vol. 10. P. 69. doi: 10.3389/fnsys.2016.00069

[9] Aldridge S. Agilent buys Her2 test firm. Nat Biotechnol. 2012;30(8):730. doi: 10.1038/nbt0812-730b

[10] Aldridge S. Agilent buys Her2 test firm // Nat Biotechnol. 2012. Vol. 30, N 8. P. 730. doi: 10.1038/nbt0812-730b

[11] Hopperton KE, Mohammad D, Trépanier MO, et al. Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review. Mol Psychiatry. 2018;23(2):177–198. doi: 10.1038/mp.2017.246

[12] Hopperton K.E., Mohammad D., Trépanier M.O., et al. Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review // Mol Psychiatry. 2018. Vol. 23. N 2. P. 177–198. doi: 10.1038/mp.2017.246

[13] Ren Y, Ao Y, O’Shea TM, et al. Ependymal cell contribution to scar formation after spinal cord injury is minimal, local and dependent on direct ependymal injury. Sci Rep. 2017;7:41122. doi: 10.1038/srep41122

[14] Ren Y., Ao Y., O’Shea T.M., et al. Ependymal cell contribution to scar formation after spinal cord injury is minimal, local and dependent on direct ependymal injury // Sci Rep. 2017. Vol. 7. P. 41122. doi: 10.1038/srep41122

[15] Taft JR, Vertes RP, Perry GW. Distribution of GFAP+ astrocytes in adult and neonatal rat brain. Int J Neurosci. 2005;115(9):1333–1343. doi: 10.1080/00207450590934570

[16] Taft J.R., Vertes R.P., Perry G.W. Distribution of GFAP+ astrocytes in adult and neonatal rat brain // Int J Neurosci. 2005. Vol. 115, N 9. P. 1333–1343. doi: 10.1080/00207450590934570

[17] Hu J, Cai D, Zhao Z, et al. Suppression of heterogeneous nuclear ribonucleoprotein c inhibit hepatocellular carcinoma proliferation, migration, and invasion via Ras/MAPK signaling pathway. Front Oncol. 2021;11:659676. doi: 10.3389/fonc.2021.659676

[18] Hu J., Cai D., Zhao Z., et al. Suppression of heterogeneous nuclear ribonucleoprotein c inhibit hepatocellular carcinoma proliferation, migration, and invasion via Ras/MAPK signaling pathway // Front Oncol. 2021. Vol. 11. P. 659676. doi: 10.3389/fonc.2021.659676

[19] Sufieva DA, Kirik OV, Alekseeva OS, Korzhevskii DE. Intermediate filament proteins in tanycytes of the third cerebral ventricle in rats during postnatal ontogenesis. Journal of Evolutionary Biochemistry and Physiology. 2016;52(6):490–498. EDN: WINOVY doi: 10.1134/S1234567816060082

[20] Суфиева Д.А., Кирик О.В., Алексеева О.С., Коржевский Д.Э. Белки промежуточных филаментов в таницитах третьего желудочка головного мозга крысы в постнатальном онтогенезе // Журнал эволюционной биохимии и физиологии. 2016. Т. 52, № 6. С. 436–443. EDN: XWQZEJ

[21] Nikitina IA, Razenkova VA, Kirik OV, Korzhevskii DE. Visualisation of Kupffer cells in the rat liver with poly- and monoclonal antibodies against microglial-specific protein Iba-1. Medical Academic Journal. 2023;23(1):85–94. EDN: UIWLVL doi: 10.17816/MAJ133649

[22] Никитина И.А., Разенкова В.А., Кирик О.В., Коржевский Д.Э. Выявление популяции клеток Купфера в печени крысы с использованием моноклональных и поликлональных антител к микроглиальному маркеру Iba-1 // Медицинский академический журнал. 2023. Т. 23, № 1. С. 85–94. EDN: UIWLVL doi: 10.17816/MAJ133649

[23] Shojaeian S, Lay NM, Zarnani AH, et al. Detection systems in immunohistochemistry. In: Streckfus CF, editor. Immunohistochemistry — the ageless biotechnology. London: IntechOpen; 2020. 140 p. doi: 10.5772/intechopen.82072

[24] Shojaeian S., Lay N.M., Zarnani A.H., et al. Detection systems in immunohistochemistry. In: Streckfus C.F., editor. Immunohistochemistry — the ageless biotechnology. London: IntechOpen, 2020. 140 p. doi: 10.5772/intechopen.82072

[25] Butler JL, Barham BJ, Heidenreich BA. Comparison of indirect peroxidase and avidin-biotin-peroxidase complex (ABC) immunohistochemical staining procedures for C-FOS in rat brain. J Anat. 2019;234(6):936–942. doi: 10.1111/joa.12967

[26] Butler J.L., Barham B.J., Heidenreich B.A. Comparison of indirect peroxidase and avidin-biotin-peroxidase complex (ABC) immunohistochemical staining procedures for C-FOS in rat brain // J Anat. 2019. Vol. 234, N 6. P. 936–942. doi: 10.1111/joa.12967

[27] Nahm M, Der-Balian GP, Venturini D, et al. Antigenic similarities of rat and mouse IgG subclasses associated with anti-carbohydrate specificities. Immunogenetics. 1980;11(2):199–203. doi: 10.1007/BF01567785

[28] Nahm M., Der-Balian G.P., Venturini D., et al. Antigenic similarities of rat and mouse IgG subclasses associated with anti-carbohydrate specificities // Immunogenetics. 1980. Vol. 11. N 2. P. 199–203. doi: 10.1007/BF01567785

[29] Kirik OV, Korzhevskiy DE. Extraependymal ependymocytes in the rat brain. Morphology. 2013;143(3):071–073. EDN: QIAGYF

[30] Кирик О.В., Коржевский Д.Э. Внеэпендимные эпендимоциты головного мозга крысы // Морфология. 2013. Т. 143, № 3. С. 071–073. EDN: QIAGYF

[31] Korzhevsky DE, Kirik OV, Petrova ES, et al. Theoretical bases and practical application of immunohistochemistry methods. 2nd edition, corrected and supplemented. Saint Petersburg: SpeczLit; 2014. (In Rus). EDN: SINOMT

[32] Коржевский Д.Э., Кирик О.В., Петрова Е.С., и др. Теоретические основы и практическое применение методов иммуногистохимии. 2-е издание, исправленное и дополненное. Санкт-Петербург: СпецЛит, 2014. EDN: SINOMT

[33] Kalinina DS, Ptukha MA, Goriainova AV, et al. Role of the trace amine associated receptor 5 (TAAR5) in the sensorimotor functions. Sci Rep. 2021;11(1):23092. doi: 10.1038/s41598-021-02289-w

[34] Kalinina D.S., Ptukha M.A., Goriainova A.V., et al. Role of the trace amine associated receptor 5 (TAAR5) in the sensorimotor functions // Sci Rep. 2021. Vol. 11, N 1. P. 23092. doi: 10.1038/s41598-021-02289-w

[35] Munguía-Martínez MF, Nava-Ruíz C, Ruíz-Díaz A, et al. Immunohistochemical study of antioxidant enzymes regulated by Nrf2 in the models of epileptic seizures (KA and PTZ). Oxid Med Cell Longev. 2019;2019:1327986. doi: 10.1155/2019/1327986

[36] Munguía-Martínez M.F., Nava-Ruíz C., Ruíz-Díaz A., et al. Immunohistochemical study of antioxidant enzymes regulated by Nrf2 in the models of epileptic seizures (KA and PTZ) // Oxid Med Cell Longev. 2019. Vol. 2019. P. 1327986. doi: 10.1155/2019/1327986

[37] Pervin M, Hasan I, Kobir MA, et al. Immunophenotypic analysis of the distribution of hepatic macrophages, lymphocytes and hepatic stellate cells in the adult rat liver. Anat Histol Embryol. 2021;50(4):736–745. doi: 10.1111/ahe.12718

[38] Pervin M., Hasan I., Kobir M.A., et al. Immunophenotypic analysis of the distribution of hepatic macrophages, lymphocytes and hepatic stellate cells in the adult rat liver // Anat Histol Embryol. 2021. Vol. 50, N 4. P. 736–745. doi: 10.1111/ahe.12718

[39] Suvarna SK, Layton C, Bancroft JD. Bancroft’s theory and practice of histological techniques. 8th ed. Elsevier; 2019.

[40] Suvarna S.K., Layton C., Bancroft J.D. Bancroft’s theory and practice of histological techniques. 8th ed. Elsevier, 2019.

Funding

Правительство РФGovernment of the Russian Federation

RIGHTS & PERMISSIONS

Eco-Vector