Summary

ऊतक और माउस संवेदी तंत्रिका फाइबर त्वचा और अंग की हड्डी Innervating तैयार Immunostaining

Published: January 26, 2012
doi:

Summary

परिधीय संवेदी तंत्रिका फाइबर subtypes के Immunocytochemical पहचान (और उसमें प्रोटीन अभिव्यक्ति का पता लगाने के) आणविक परिधीय सनसनी अंतर्निहित तंत्र को समझने के लिए महत्वपूर्ण हैं. यहाँ हम परिधीय / आंत जैसे त्वचा और अंग हड्डियों के ऊतकों के नमूनों, परिधीय संवेदी तंत्रिका तंतुओं के विशिष्ट immunostaining के लिए, की तैयारी के लिए विधियों का वर्णन.

Abstract

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Protocol

1. पशु परफ्यूज़न सभी पशु इस अध्ययन में प्रदर्शन प्रक्रियाओं संस्थागत पशु देखभाल और आयोवा विश्वविद्यालय का प्रयोग समिति द्वारा अनुमोदित कर रहे हैं, और अनुसंधान में पशुओं के उपयोग के लिए NIH दिश…

Discussion

यहाँ हम माउस त्वचा और हड्डी immunostaining और परिधीय संवेदी तंत्रिका तंतुओं का पता लगाने के लिए ऊतक वर्गों की तैयारी के लिए तरीकों की विस्तृत है. तल पंच बायोप्सी से उत्पादित वर्गों दोनों अलोम और बालों त्वचा के ह…

Declarações

The authors have nothing to disclose.

Acknowledgements

हम उनकी मदद के लिए confocal माइक्रोस्कोपी / माउस तल पंच बायोप्सी immunostaining की इमेजिंग के प्रारंभिक मानकीकरण में डा. यूरी एम. Usachev धन्यवाद, और उसे जारी की मदद और इस काम में रचनात्मक आलोचना के लिए डॉ. डोना एल हैमंड. इस काम NINDS NIH / (NS069898) से अनुदान, और रक्षा प्रोस्टेट कैंसर रिसर्च प्रोग्राम के विभाग (DoD – PCRP 101,096) से एक आइडिया विकास अनुदान DPM पुरस्कार द्वारा वित्त पोषित किया गया था

Materials

Material Name Tipo Company Catalogue No. Comment
3mm Harris Micro-Punch Material Ted Pella 15094  
Perfusion pump Material VWR International 23609-170  
Paraformaldehyde Reagent Fisher Scientific T353  
Picric acid Reagent Sigma-Aldrich 239801  
OCT Embedding compound Reagent Tissue-Tek 4583  
Cyto-Freeze cryogenic aerosol spray Material Control Company 3118  
Goat Serum Reagent Sigma-Aldrich G9023  
Incubation tray and lid for Immunostaining (Large) Material RPI Corp. 248270 (tray)
248270-A (lid)
 
ImmEdge hydrophobic barrier pen Material Vector Laboratories H-4000  
Camel’s Hair Brushes (#1 thickness) Material Ted Pella 11859  
Pro‐Long Gold Mounting medium Reagent Invitrogen P36930  

Referências

  1. Hoffman, E. M., Schechter, R., Miller, K. E. Fixative Composition Alters Distributions of Immunoreactivity for Glutaminase and Two Markers of Nociceptive Neurons, Nav 1.8 and TRPV1, in the Rat Dorsal Root Ganglion. Journal of Histochemistry & Cytochemistry. 58, 329-344 (2010).
  2. Neves, J. D. S., Omar, N. F., Narvaes, E. A. O., Gomes, J. R., Novaes, P. D. Influence of different decalcifying agents on EGF and EGFR immunostaining. Acta Histochemica. 113, 484-488 (2011).
  3. Watson, R. E., Wiegand, S. J., Clough, R. W., Hoffman, G. E. Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology. Peptides. 7, 155-159 (1986).
  4. Yen, L. D., Bennett, G. J., Ribeiro-da-Silva, A. Sympathetic sprouting and changes in nociceptive sensory innervation in the glabrous skin of the rat hind paw following partial peripheral nerve injury. The Journal of Comparative Neurology. 495, 679-690 (2006).
  5. Boyette-Davis, J., Xin, W., Zhang, H., Dougherty, P. M. Intraepidermal nerve fiber loss corresponds to the development of Taxol-induced hyperalgesia and can be prevented by treatment with minocycline. Pain. 152, 308-313 (2011).
  6. Bloom, A. P. Breast Cancer-Induced Bone Remodeling, Skeletal Pain, and Sprouting of Sensory Nerve Fibers. The Journal of Pain. 12, 698-711 (2011).
  7. Constantin, C. E. Endogenous Tumor Necrosis Factor α (TNFα) Requires TNF Receptor Type 2 to Generate Heat Hyperalgesia in a Mouse Cancer Model. J. Neurosci. 28, 5072-5081 (2008).
  8. Jankowski, M. P. Sensitization of Cutaneous Nociceptors after Nerve Transection and Regeneration: Possible Role of Target-Derived Neurotrophic Factor Signaling. The Journal of Neuroscience. 29, 1636-1647 (2009).
  9. Jimenez-Andrade, J. M. Pathological Sprouting of Adult Nociceptors in Chronic Prostate Cancer-Induced Bone Pain. J. Neurosci. 30, 14649-14656 (2010).
  10. Persson, A. -. K. Sodium-calcium exchanger and multiple sodium channel isoforms in intra-epidermal nerve terminals. Molecular Pain. 6, 84-84 (2010).
  11. Ohshima, M., Miyake, M., Takeda, M., Kamijima, M., Sakamoto, T. Staphylococcal Enterotoxin B Causes Proliferation of Sensory C-Fibers and Subsequent Enhancement of Neurogenic Inflammation in Rat Skin. Journal of Infectious Diseases. 203, 862-869 (2011).
  12. Johnson, M. S., Ryals, J. M., Wright, D. E. Early loss of peptidergic intraepidermal nerve fibers in an STZ-induced mouse model of insensate diabetic neuropathy. Pain. 140, 35-47 (2008).
check_url/pt/3485?article_type=t

Play Video

Citar este artigo
Shepherd, A. J., Mohapatra, D. P. Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones. J. Vis. Exp. (59), e3485, doi:10.3791/3485 (2012).

View Video