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: 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