Journal: The Journal of Physiological Sciences : JPS

Article Title: Immobilization-induced hypersensitivity associated with spinal cord sensitization during cast immobilization and after cast removal in rats

doi: 10.1007/s12576-013-0277-4

Figure Lengend Snippet: Intensity of calcitonin gene-related peptide (CGRP) expression in the ipsilateral dorsal horn of the spinal cord. Representative photomicrographs of CGRP immunohistochemistry in the ipsilateral dorsal horn from the Im-4 weeks, Im-8 weeks, and the control groups (age-matched with the Im-4 weeks group) are shown (a). The CGRP-positive neural fibers were clearly observed in the deep layer of the dorsal horn only in the Im-8 weeks group (arrowheads). Percentage control of fluorescence intensity of CGRP expression in the superficial layer (laminae I-II) (b, d) and deep layers (laminae III-VI) were calculated (c, e) in the ipsilateral and contralateral dorsal horn. *P < 0.05, significantly different compared to the age-matched control group. # P < 0.05, significantly different compared to the Im-4 weeks group. Scale bar = 100 μm

Article Snippet: Next, sections were blocked for 20 min with 5 % bovine albumin dissolved in PBS, followed by incubation with an anti-CGRP polyclonal antibody (1:500 rabbit; ImmunoStar Inc. Hudson, WI, USA) overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Control, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The leukotriene B4 receptors BLT1 and BLT2 form an antagonistic sensitizing system in peripheral sensory neurons

doi: 10.1074/jbc.M116.769125

Figure Lengend Snippet: BLT1 and BLT2 are expressed in sensory neurons. A and B, representative images showing the colocalization of BLT1 and BLT2 with IB4 (A) and CGRP (B) in dorsal root ganglia. The white bar represents 30 μm. Arrows depict cells expressing LTB4 receptors.

Article Snippet: Anti-BLT1 and anti-BLT2 antibodies were from Hölzel Diagnostika (Köln, Germany), anti-TRPV1 antibody was from Alomone Labs, FITC-labeled IB4 was from Sigma-Aldrich, and anti-cGRP antibody was from Alpha Diagnostics International Inc.

Techniques: Expressing

Journal: Molecular Pain

Article Title: The majority of dorsal spinal cord gastrin releasing peptide is synthesized locally whereas neuromedin B is highly expressed in pain- and itch-sensing somatosensory neurons

doi: 10.1186/1744-8069-8-52

Figure Lengend Snippet: Characterizing the localization of Grp in DRG, spinal cord, and dorsal root using anti-bombesin antiserum. ( A - B ) DRGs immunostained with antibodies against bombesin (red) and Cgrp (green). Arrow indicates a bombesin + ;Cgrp + neuron. ( C - D ) DRG neurons in adjacent sections are immunostained with anti-bombesin antiserum preabsorbed with purified Grp (red) and anti-Cgrp antibody (green). Preabsorption results in a complete loss of anti-bombesin immunoreactivity. ( E - F ) DRG neurons immunostained with antibodies against bombesin (red) and Pap (green). ( G - H ) DRG neurons immunostained with anti-bombesin anti-serum (red) and fluorescently-conjugated IB4 (green). ( I - J ) DRG neurons immunostained with antibodies against bombesin (red) and VGlut1 (green). Arrows in ( E-J ) indicate bombesin + neurons that are not immunopositive for the costaining marker. ( K - L ) dSC section immunostained with antibodies against bombesin (red) and Cgrp (green). The dSC layers positive for bombesin are outlined with white dots and higher magnification of the staining is shown in the inset. ( M - N ) dSC section immunostained with anti-bombesin antiserum preabsorbed with purified Grp (red) and anti-Cgrp antibody (green), showing a complete loss of anti-bombesin immunoreactivity. ( O - P ) dSC section immunostained with antibodies against bombesin (red) and Pap (green). ( Q - R ) dSC section immunostained with anti-bombesin serum (red) and fluorescently-conjugated IB4 (green). ( S - T ) dSC section immunostained with antibodies against bombesin (red) and VGlut1 (green). ( U ) A high magnification confocal image of adult dSC immunostained with anti-bombesin (red) and anti-NeuN (green). ( V - X ) A higher magnification of wild type adult dorsal root immunostained with antibodies against bombesin (red) and Cgrp (green) reveals the presence of Cgrp, but not Grp, in axons projecting from the DRG to dSC. Dorsal root is outlined by dotted line. Arrow indicates the dSC anti-bombesin immunoreactivity. Scale bars = 50 μm ( A-J ), 100 μm ( K-T ), 5 μm ( U ), 30 μm ( V-X ).

Article Snippet: Anti-bombesin and anti-Cgrp antibodies at the concentrations described above were added to PBS containing 5% lamb serum, 0.3% TritonX-100, and bombesin peptide (50 μg/mL; American Peptide Company, 16-7-10A) or GRP peptide (50 μg/mL; American Peptide Company, 62-3-10) and incubated O/N at 4°C with gentle agitation.

Techniques: Purification, Marker, Staining

Journal: Molecular Pain

Article Title: The majority of dorsal spinal cord gastrin releasing peptide is synthesized locally whereas neuromedin B is highly expressed in pain- and itch-sensing somatosensory neurons

doi: 10.1186/1744-8069-8-52

Figure Lengend Snippet: Dorsal root rhizotomy causes a dramatic loss of Pap and Cgrp but significantly smaller changes of bombesin and Pkcγ in dSC. L4, L5, and L6 central roots of two month old wild type mice were transected, and mice were examined two weeks following surgery. Sections of the lumbar enlargement were immunostained to examine the loss of proteins and peptides caused by dorsal root axonal degeneration and the contralateral side of the same spinal cord section was used as the control. ( A - F ) Sections were immunostained with antibodies against bombesin (red) and Pkcγ (green), a protein highly synthesized in dSC neurons (control side (A-C) and transected side (D-F)). Comparable and modest loss of both bombesin and Pkcγ immunoreactivity are found after the dorsal root rhizotomy. Enhanced green fluorescence and abnormal morphology of the transected dorsal root were also noted, which may be caused by axonal inflammation after injury. ( G - L ) Sections immunostained with antibodies against bombesin (red) and Pap (green). As noted, dSC Pap is dramatically decreased after the dorsal root rhizotomy. ( M - R ) Sections immunostained with antibodies against bombesin (red) and Cgrp (green), another marker derived from DRG neurons. Similar to Pap, dSC Cgrp is also greatly reduced following the dorsal root rhizotomy. Scale bar = 100 μm. ( S ) Quantification and statistical analysis of dynamic changes of dSC Grp, Pkcγ, Pap, and Cgrp following the dorsal root rhizotomy. The loss of dSC Grp after dorsal root rhizotomy is significantly different from those of Pap and Cgrp (P < 0.01), and the same is true for dSC Pkcγ. In contrast, no significant differences are found between Grp and Pkcγ (P = 0.44) or Pap and Cgrp (P = 0.25).

Article Snippet: Anti-bombesin and anti-Cgrp antibodies at the concentrations described above were added to PBS containing 5% lamb serum, 0.3% TritonX-100, and bombesin peptide (50 μg/mL; American Peptide Company, 16-7-10A) or GRP peptide (50 μg/mL; American Peptide Company, 62-3-10) and incubated O/N at 4°C with gentle agitation.

Techniques: Synthesized, Fluorescence, Marker, Derivative Assay