Comparison of formaldehyde and methanol fixatives used in the detection of ion channel proteins in isolated rat ventricular myocytes by immunofluorescence labelling and confocal microscopy

• Autorzy: Yamanushi T.T. , Boyett M.R. , Yamamoto Y. , Ohsaki H. , Hirakawa E. , Dobrzynski H.
• Języki publikacji: EN

Abstrakty

EN

In this study, a fixation protocol using a 10% neutral buffered formalin (FA) solution and another protocol using a methanol (MeOH) solution were compared for detection of ion channels, Kᵥ 1.5, Kᵥ 4.2, Caᵥ 1.2, Kᵢᵣ6.2, Naᵥ 1.5 and Naᵥ 1.1 in rat myocytes by immunolabelling. Kᵥ 1.5 and Kᵥ 4.2 at intercalated discs and Caᵥ 1.2 at transverse tubules were not detected by FA but were detected by MeOH. Kᵢᵣ6.2 at transverse tubules and Naᵥ 1.5 at sarcolemma were detected by FA but not by MeOH. It is suggested that both FA and MeOH fixation protocols should be used for the detection of cardiac ion channels by immunolabelling. (Folia Morphol 2015; 74, 2: 258–261)

• Wydawca: -
• Czasopismo: Folia Morphologica
• Rocznik: 2015
• Tom: 74
• Numer: 2
• Opis fizyczny: p.258-261,fig.,ref.

Twórcy

autor

Yamanushi T.T.

• Kagawa Prefectural University of Health Sciences, 281-1 Hara, Mure-cho, Takamatsu City, Kagawa, Japan

autor

Boyett M.R.

• Cardiovascular Medicine, Faculty of Medical and Human Sciences, University of Manchester, Manchester, U.K.

autor

Yamamoto Y.

• Kagawa Prefectural University of Health Sciences, 281-1 Hara, Mure-cho, Takamatsu City, Kagawa, Japan
• Human Health Sciences, Faculty of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan

autor

Ohsaki H.

• Kagawa Prefectural University of Health Sciences, 281-1 Hara, Mure-cho, Takamatsu City, Kagawa, Japan
• Department of Medical Technology, Faculty of Health Sciences, Ehime Prefectural University of Health Sciences, 543 Takoda, Tobe-cho, Iyo-gun, Ehime, Japan

autor

Hirakawa E.

• Kagawa Prefectural University of Health Sciences, 281-1 Hara, Mure-cho, Takamatsu City, Kagawa, Japan

autor

Dobrzynski H.

• Cardiovascular Medicine, Faculty of Medical and Human Sciences, University of Manchester, Manchester, U.K.

Bibliografia

• 1. Barry DM, Trimmer JS, Merlie JP, Nerbonne JM (1995) Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels? Circ Res, 77: 361–369.
• 2. Dobrzynski H, Marples DD, Musa H, Yamanushi TT, Henderson Z, Takagishi Y, Honjo H, Kodama I, Boyett MR (2001) Distribution of the muscarinic K+ channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart. J Histochem Cytochem, 49: 1221–1234.
• 3. Dobrzynski H, Rothery SM, Marples DD, Coppen SR, Takagishi Y, Honjo H, Tamkun MM, Henderson Z, Kodama I, Severs NJ, Boyett MR (2000) Presence of the Kv1.5 K(+) channel in the sinoatrial node. J Histochem Cytochem. 48: 769–780.
• 4. Farmilo AJ, Ronald H (2001) Fixation. In: Boenisch T ed. Handbook Immunochemical staining methods. 3rd Ed. Dako Corporation, California, pp. 18–22.
• 5. French D, Edsall JH (1945) The reaction of formaldehyde with amino acids and proteins. Adv Protein Chemi, 2: 277–335.
• 6. Jamur MC, Oliver C (2010) Cell fixatives for immunostaining. Methods Mol Biol, 588: 55–61.
• 7. Lojda Z (1965) Fixation in histochemistry. Folia Morphol, 13: 65–83.
• 8. Maier SK, Westenbroek RE, Schenkman KA, Feigl EO, Scheuer T, Catterall WA (2002) An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart. P Natl Acad Sci USA, 99: 4073–4078.
• 9. Musa H, Lei M, Honjo H, Jones SA, Dobrzynski H, Lancaster MK, Takagishi Y, Henderson Z, Kodama I, Boyett MR (2002) Heterogeneous expression of Ca(2+) handling proteins in rabbit sinoatrial node. J Histochem Cytochem, 50: 311–324.
• 10. Opie LH (2004) Heart physiology from cell to circulation. 4th Ed. Lippincott Williams & Wilkins, Philadelphia.
• 11. Seharaseyon J, Ohler A, Sasaki N, Fraser H, Sato T, Johns DC, O’Rourke B, Marban E (2000) Molecular composition of mitochondrial ATP-sensitive potassium channels probed by viral Kir gene transfer. J Mol Cell Cardiol, 32: 1923–1930.
• 12. Shui Z, Boyett MR, Zang WJ (1997) ATP-dependent desensitization of the muscarinic K+ channel in rat atrial cells. J Physiol, 505: 77–93.
• 13. Smith-Clerc J, Hinz B (2010) Immunofluorescence detection of the cytoskeleton and extracellular matrix in tissue and cultured cells. Methods Mol Biol, 611: 43–57.
• 14. Yamanushi TT, Shui Z, Leach RN, Dobrzynski H, Claydon TW, Boyett MR (2007) Role of internalization of M2 muscarinic receptor via clathrin-coated vesicles in desensitization of the muscarinic K+ current in heart. Am J Physiol Heart Circ Physiol, 292: H1737–H1746.
• 15. Yoo S, Dobrzynski H, Fedorov VV, Xu SZ, Yamanushi TT, Jones SA, Yamamoto M, Nikolski VP, Efimov IR, Boyett MR (2006) Localization of Na+ channel isoforms at the atrioventricular junction and atrioventricular node in the rat. Circulation, 114: 1360–1371.

Identyfikatory

DOI

10.5603/FM.2015.0041