Journal: Brain Sciences

Article Title: Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors

doi: 10.3390/brainsci10060344

Figure Lengend Snippet: Expression of Ca V 2 high voltage activated calcium channels. ( A – C ) D50 iDNs showed robust expression of all Ca V 2 high voltage activated calcium channels in regions of the soma, neurites, as well as axonal varicosities. Arrowheads indicate individual ( A ) Ca V 2.1/PQ-type, ( B ) Ca V 2.2/N-type, and ( C ) Ca V 2.3/R-type clusters along neurite structures of iDNs. Line scan measurements for individual Ca V 2 clusters on TUJ1-IR+ varicosities revealed their location within putative synaptic structures in iDN cultures, line scan was set to 3 µm. ( D – F ) Mouse neurons (mDRG) served as controls, line scans performed on neurite segments, no axonal varicosities were detected, 2–3 individual differentiations, scale bars 20 µm (overview, soma), 10 µm (enlargement, axon), 1 µm (axonal varicosities, putative synapses).

Article Snippet: Primary antibodies used were anti-synapsin (1:1000, mouse monoclonal, Synaptic Systems (SySy); #106011 reference [ ]); anti-BRN3A (1:500 rabbit polyclonal, SySy, #411003, reference [ ]); anti-ISL-1 (1:500, rabbit polyclonal, SySy, #406003 reference [ ]); anti-Ca V 2.1 (1:1000, rabbit polyclonal, SySy, #152103 k.o verified); anti-Ca V 2.2 (1:1000, rabbit polyclonal, SySy, #152313 reference [ ]); anti-Ca V 2.3 (1:1000, rabbit polyclonal, SySy, #152403 reference [ ]); anti-RUNX1 (1:200, rabbit polyclonal, Abcam, #23980 reference [ ]); anti-p75 (1:200, rabbit polyclonal, Abcam, #AB52987 reference [ ]); anti-NKCC3 (1:500, rabbit polyclonal, Thermo Fisher Scientific #PA5-56975); anti-TRPV1 (1:200, rabbit polyclonal, Alomone Labs, #ACC-030 reference [ ]); anti-TUJ1 (1:600, mouse monoclonal, R&D Systems, #MAB1195 reference [ ]); anti-GABA A R (β2,3 chain, representing the most abundant subunit chains of GABA A Rs) (1:1000, mouse monoclonal, Chemicon, #MAB341 reference [ ]); and anti-HNC1–4 (1:600, Alomone Labs, HCN1 #APC-056, HCN2 #APC-030, HCN3 #APC-057, and HCN4 #APC-052, reference [ ]).

Techniques: Expressing

Journal: Gene therapy

Article Title: AAV-encoded Ca V 2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain

doi: 10.1038/s41434-019-0082-7

Figure Lengend Snippet: CaV2.2α1b protein expression is significantly increased in the lumbar DRG following TNI. Immunoblot reveals a clean band around ~230 KDa of CaV2.2α1b protein in the homogenate of rat brain cortex (a, left panel), and preincubation with excess immunogenic peptide completely eliminated this band (a, right panel). The NKA1α-deficient cytosolic fraction and NKA1α-enriched plasma membrane (PM) fraction were extracted from the DRG tissues at 4 weeks after TNI, and subjected to immunoblotting as shown in the representative immunoblots of CaV2.2α1b, CRMP2, Tubb3, and NKA1α of cytosol (b) and PM fractions (c), respectively. Bar charts (d) show densitometry analysis of immunoblots. The number in each bar is the number of analyzed DRG per group. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively (Two tailed unpaired Student’s t-test). Representative montage images show immunostaining of CaV2.2α1b (green) and colabeling with NKA1α (red) in the merged image from the DRG sections of control (e, f) and TNI rat (g, h). Scale bar: 100 μm for all IHC images

Article Snippet: The specificity of the rabbit polyclonal Ca V 2.2α1b antibody (Atlas Antibodies, Stockholm, Sweden), which is raised against a synthetic peptide corresponding to human Ca V 2.2α1b (amino acids 1857–1950 that has 94% identity to rat Ca V 2.2α1b sequence) has been validated showing identification of neuronal Ca V 2.2α1b immunopositive profile in DRG by a previous publication [ 83 ].

Techniques: Expressing, Western Blot, Two Tailed Test, Immunostaining

Journal: Gene therapy

Article Title: AAV-encoded Ca V 2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain

doi: 10.1038/s41434-019-0082-7

Figure Lengend Snippet: AAV6-CBD3A6K treatment reverses increased CaV2.2α1b protein level in DRG. Representative montage IHC images show profiles of immunolabeling of CaV2.2α1b (a), EGFP-tagged CBD3A6K 6 weeks after AAV6-CDD3A6K treatment (b), Hoechst nuclear staining (c), and colabeling of CaV2.2α1b and EGFP-tagged CBD3A6K in the merged image (d) on a ipsilateral L5 DRG section from TNI rat. EGFP-tagged CBD3A6K distributes in the afferent central terminals of the TNI ipsilateral DH (e) with abolished IB4 staining in the tibial nerve central terminal zone (arrowheads). Western blots verifies transgenes (EGFP and EGFP-CBD3A6K) expression in DRG 6 weeks after vector injection, showing EGFP-CBD3A6K band ~5 KDa larger than EGFP (f). Representative immunoblots of CaV2.2α1b, CRMP2, NKA1α, and Tubb3 on the cytosol (g) and PM fractions (h) extracted from control and TNI only DRG, as well as the TNI DRG after vector treatment, as indicated, respectively. Bar charts (i) show densitometry analysis of immunoblots, *p < 0.05, **p < 0.01, and ***p < 0.001 for comparison of CaV2.2α1b and CRMP2 protein level in the cytosol and PM fractions in each group (One-way ANOVA analysis of variance with Turkey post-hoc test). The number in each bar is the number of analyzed DRG per group. Scale bar: 100 μm for all IHC images

Article Snippet: The specificity of the rabbit polyclonal Ca V 2.2α1b antibody (Atlas Antibodies, Stockholm, Sweden), which is raised against a synthetic peptide corresponding to human Ca V 2.2α1b (amino acids 1857–1950 that has 94% identity to rat Ca V 2.2α1b sequence) has been validated showing identification of neuronal Ca V 2.2α1b immunopositive profile in DRG by a previous publication [ 83 ].

Techniques: Immunolabeling, Staining, Western Blot, Expressing, Plasmid Preparation, Injection

Journal: Gene therapy

Article Title: AAV-encoded Ca V 2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain

doi: 10.1038/s41434-019-0082-7

Figure Lengend Snippet: CaV2.2α1b protein expression is significantly increased in the lumbar DH following TNI. PKCγ colabeling with CGRP (a) or IB4 (b) followed by ImageJ line scan analysis (c) delineates the profiles and length of superficial DH (laminae I and II) and deeper layers of the DH (laminae III and IV). CaV2.2α1b immunoreactive (IR) staining (red) in the DH of control animal shows symmetrical intensity (d), which is confirmed by line scans (f). In contrast, CaV2.2α1b-IR intensity in the ipsilateral (ipsi.) DH of TNI rats appears increased compared to contralateral (contra.) side (e), which is verified by line scans (g). The effect of TNI is further confirmed by analysis of the ratio of peak intensities (ipsi./contra, h) (Two-tailed unpaired Student’s t-test). Numbers in each bar represents the spinal cords analyzed from each group. In parallel, control spinal DH shows symmetrical immunostaining intensity of the CaV2.2 auxiliary subunit α2δ1 (CaV2.2α2δ1), while the CaV2.2α2δ1 level is apparently increased on the ipsilateral side compared to contralateral side in the TNI rat. Scale bar: 100 μm for all IHC images. ** p < 0.01

Article Snippet: The specificity of the rabbit polyclonal Ca V 2.2α1b antibody (Atlas Antibodies, Stockholm, Sweden), which is raised against a synthetic peptide corresponding to human Ca V 2.2α1b (amino acids 1857–1950 that has 94% identity to rat Ca V 2.2α1b sequence) has been validated showing identification of neuronal Ca V 2.2α1b immunopositive profile in DRG by a previous publication [ 83 ].

Techniques: Expressing, Staining, Two Tailed Test, Immunostaining

Journal: Gene therapy

Article Title: AAV-encoded Ca V 2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain

doi: 10.1038/s41434-019-0082-7

Figure Lengend Snippet: AAV6-CBD3A6K treatment normalizes CaV2.2α1b upregulation in the DH. Representative images show profiles of immunolabeling of CaV2.2α1b in the DH from control (a) and TNI only (b), control colabeled CaV2.2α1b (c) with EGFP (c1) after AAV6-EGFP injection, TNI colabeled CaV2.2α1b (d) with EGFP (d1) after AAV6-EGFP injection, and TNI colabeled CaV2.2α1b (e) with EGFP (e1) after AAV6-CBD3A6K injection. Comparison of the ratios of ipsi-lateral/contralateral peak intensity (au) in control, TNI only, TNI with AAV6-EGFP injection, and TNI with AAV6-CBD3A6K treatment by ImageJ line scan function was analyzed (f). *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001, respectively (One-way ANOVA with Turkey post-hoc analysis). Scale bar: 100 μm for all IHC images

Article Snippet: The specificity of the rabbit polyclonal Ca V 2.2α1b antibody (Atlas Antibodies, Stockholm, Sweden), which is raised against a synthetic peptide corresponding to human Ca V 2.2α1b (amino acids 1857–1950 that has 94% identity to rat Ca V 2.2α1b sequence) has been validated showing identification of neuronal Ca V 2.2α1b immunopositive profile in DRG by a previous publication [ 83 ].

Techniques: Immunolabeling, Injection

Journal: Gene therapy

Article Title: AAV-encoded Ca V 2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain

doi: 10.1038/s41434-019-0082-7

Figure Lengend Snippet: Primary antibodies and IgG controls used in this study

Article Snippet: The specificity of the rabbit polyclonal Ca V 2.2α1b antibody (Atlas Antibodies, Stockholm, Sweden), which is raised against a synthetic peptide corresponding to human Ca V 2.2α1b (amino acids 1857–1950 that has 94% identity to rat Ca V 2.2α1b sequence) has been validated showing identification of neuronal Ca V 2.2α1b immunopositive profile in DRG by a previous publication [ 83 ].

Techniques:

Journal: Journal of Ovarian Research

Article Title: Expression of voltage-gated Ca 2+ channels, Insp 3 Rs, and RyRs in the immature mouse ovary

doi: 10.1186/s13048-022-01015-y

Figure Lengend Snippet: Quantification of specific immunolabelling of voltage-gated and intracellular release Ca 2+ channels in the neonatal (left column), early infantile (middle column), and late infantile (right column) ovary. The fluorescence intensity of voltage-gated Ca V 1.2 ( A , A 1 , A 2 ), Ca V 1.3 ( B , B 1 , B 2 ), Ca V 2.1 ( C , C 1 , C 2 ), and Ca V 2.2 ( D, D 1 , D 2 ), as well as InsP 3 R ( E , E 1 , E 2 ) and RyR ( F , F 1 , F 2 ) from the different ovarian structures (primordial, primary, and secondary oocytes, granulosa cells, theca cells, and interfollicular stroma), was measured. The mean fluorescence intensities (arbitrary units) were plotted as bar graphs. A-F : PND3. A 1 -F 1 : PND8. A 2 -F 2 : PND16. The standard error of the mean of each bar is indicated and the number of objects measured of each type. The differences between bars are statistically significant ( p <0.05), except for a few bars in Figures A1, D1, and F1

Article Snippet: Then, sections were incubated for 24 hrs. in a humid chamber at 4°C with one of the primary rabbit antibodies from Alomone Labs (Jerusalem, Israel): anti-Ca V 2.1 ( α 1A; validation number 2039764, lot # AN-09), anti-Ca V 2.2 ( α 1B; validation number 2039766, lot # AN-015), anti-Ca V 1.2 ( α 1C; validation number 2039771, lot # AN-19) or anti-Ca V 1.3 ( α 1D; validation number 2039775, lot # AN-09 : dilution 1:100), rabbit anti-InsP 3 R (I, II, III) H-300 Santa Cruz Biotechnologies (sc-28613; dilution 1:50) and mouse anti-RyR C3-33, (ab2827; dilution 1:20) from ABCAM.

Techniques: Fluorescence

Journal: Journal of Ovarian Research

Article Title: Expression of voltage-gated Ca 2+ channels, Insp 3 Rs, and RyRs in the immature mouse ovary

doi: 10.1186/s13048-022-01015-y

Figure Lengend Snippet: Tissue distribution of specific immunolabelling for Ca V 2.1 and Ca V 2.2 voltage-gated Ca 2+ channels in the neonatal ovary (PND 3). A: Ca V 2.1 specific immunostaining. a , b: Enlarged images from the square areas indicated in A . yellow and red asterisks: Strong staining of primordial and primary follicles, respectively. yellow arrows: Ca V 2.1 positive granulosa cells surrounding primary follicles. blue arrows: Intensively Ca V 2.1 immunoreactive cells (possibly autonomic neurons). blue asterisks: unstained stromal cells. B: Ca V 2.2 specific immunostaining. c , d: Enlarged images from the square areas indicated in B . yellow and red asterisks: Strong Ca V 2.2 immunostaining of primordial and primary follicles, respectively. yellow arrows: positive granulosa cells surrounding primary follicles blue asterisks: unstained stromal cells. Calibration bar: 100 μm (A, B); 50 μm (a, b, c, d)

Article Snippet: Then, sections were incubated for 24 hrs. in a humid chamber at 4°C with one of the primary rabbit antibodies from Alomone Labs (Jerusalem, Israel): anti-Ca V 2.1 ( α 1A; validation number 2039764, lot # AN-09), anti-Ca V 2.2 ( α 1B; validation number 2039766, lot # AN-015), anti-Ca V 1.2 ( α 1C; validation number 2039771, lot # AN-19) or anti-Ca V 1.3 ( α 1D; validation number 2039775, lot # AN-09 : dilution 1:100), rabbit anti-InsP 3 R (I, II, III) H-300 Santa Cruz Biotechnologies (sc-28613; dilution 1:50) and mouse anti-RyR C3-33, (ab2827; dilution 1:20) from ABCAM.

Techniques: Immunostaining, Staining

Journal: Journal of Ovarian Research

Article Title: Expression of voltage-gated Ca 2+ channels, Insp 3 Rs, and RyRs in the immature mouse ovary

doi: 10.1186/s13048-022-01015-y

Figure Lengend Snippet: Tissue distribution of specific immunolabelling for Ca V 2.1 and Ca V 2.2 voltage-gated Ca 2+ channels in the early infantile ovary (PND 8) . A: Ca V 2.1 specific immunostaining. a , b: Enlarged images from the square areas indicated in A . yellow asterisks: weak staining of few primordial oocytes near the plasma membrane. red asterisks: distinct Ca V 2.1 staining of oocytes and granulosa cells from primary and secondary follicles. blue arrows: cytoplasmic staining of granulosa cells close to the plasma membrane in primary follicles. yellow arrows: flat, perifollicular stromal cells next to secondary and early antral follicles show the strongest Ca V 2.1 immunolabeling. B: Ca V 2.2 specific immunostaining. c , d: Enlarged images from the square areas indicated in B . Ca V 2.2-specific staining is moderate throughout the ovary. yellow asterisks: few remaining primordial follicles . red asterisks: oocytes showing moderate Ca V 2.2 immunostaining: blue arrows: weakly stained granulosa cells surrounding oocytes from primary follicles. yellow arrows: patches of perifollicular cells around secondary and early antral follicles show the strongest Ca V 2.2 labeling. Calibration bar: 100 μm (A, B); 50 μm (a, b, c, d)

Article Snippet: Then, sections were incubated for 24 hrs. in a humid chamber at 4°C with one of the primary rabbit antibodies from Alomone Labs (Jerusalem, Israel): anti-Ca V 2.1 ( α 1A; validation number 2039764, lot # AN-09), anti-Ca V 2.2 ( α 1B; validation number 2039766, lot # AN-015), anti-Ca V 1.2 ( α 1C; validation number 2039771, lot # AN-19) or anti-Ca V 1.3 ( α 1D; validation number 2039775, lot # AN-09 : dilution 1:100), rabbit anti-InsP 3 R (I, II, III) H-300 Santa Cruz Biotechnologies (sc-28613; dilution 1:50) and mouse anti-RyR C3-33, (ab2827; dilution 1:20) from ABCAM.

Techniques: Immunostaining, Staining, Immunolabeling, Labeling

Journal: Journal of Ovarian Research

Article Title: Expression of voltage-gated Ca 2+ channels, Insp 3 Rs, and RyRs in the immature mouse ovary

doi: 10.1186/s13048-022-01015-y

Figure Lengend Snippet: Tissue distribution of specific immunolabelling for Ca V 2.1 and Ca V 2.2 voltage-gated Ca 2+ channels in the late infantile ovary (PND 16). A: Ca V 2.1 specific immunostaining. a , b: Enlarged images from the square areas indicated in A. red asterisks: oocytes and granulosa cells from primary, secondary, pre-antral, and antral follicles are express Ca V 2.1 weakly. blue arrows: in contrast, patches of perifollicular cells forming incomplete envelopes around secondary, early antral, and antral follicles are strongly stained. B: Ca V 2.2 specific immunostaining. c , d: Enlarged images from the square areas indicated in B. Weak to moderate Ca V 2.2 labeling is visible throughout the ovary. red asterisks: weakly stained granulosa cells. yellow arrows: most oocytes are weakly stained. green arrows : some oocytes display patches of intense labeling. blue arrows: patches of strongly Ca V 2.2 positive perifollicular cells form an incomplete envelope around early antral and antral follicles. yellow asterisks strongly Ca V 2.2 positive bundles of smooth muscle cells seen at the hilum. Calibration bar: 100 μm (A, B); 50 μm (a, b, c, d)

Article Snippet: Then, sections were incubated for 24 hrs. in a humid chamber at 4°C with one of the primary rabbit antibodies from Alomone Labs (Jerusalem, Israel): anti-Ca V 2.1 ( α 1A; validation number 2039764, lot # AN-09), anti-Ca V 2.2 ( α 1B; validation number 2039766, lot # AN-015), anti-Ca V 1.2 ( α 1C; validation number 2039771, lot # AN-19) or anti-Ca V 1.3 ( α 1D; validation number 2039775, lot # AN-09 : dilution 1:100), rabbit anti-InsP 3 R (I, II, III) H-300 Santa Cruz Biotechnologies (sc-28613; dilution 1:50) and mouse anti-RyR C3-33, (ab2827; dilution 1:20) from ABCAM.

Techniques: Immunostaining, Staining, Labeling