Journal: The Journal of Neuroscience

Article Title: A Syntaxin 1, Gα o , and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

doi: 10.1523/JNEUROSCI.0346-04.2004

Figure Lengend Snippet: Intensity correlation analysis, experimental models. Each panel in A, C, and E shows a calyx nerve terminal stained with two antibodies with the overlay on the left and the individual B/W image pairs on the right. Each panel in B, D, and F shows intensity correlation plots on the left together with the respective plots of stain A (center) and B (right) pixel intensities against their (A - a)(B - b) values (see Results, Intensity correlation analysis). A, B, Experimental example of dependent staining: calyx nerve terminal stained for SV2 with monoclonal (red, left panel; B/W center panel) and polyclonal (green, left panel; B/W right panel) tagged antibodies. ICQ = 0.48, psign test < 0.001. C,D, Experimental example of segregated staining. Calyx terminal stained for SV2 (red, left panel; B/W center panel) and Na/K pump (green, left panel; B/W right panel). ICQ = -0.19, psign test < 0.001. E, F, Experimental example of complex staining. A calyx nerve terminal stained with a mAb against SV2 (red, left panel; B/W center panel) and polyclonal antibody against neurofilament (green, left panel; B/W right panel). Scale bars, 5 μm.

Article Snippet: Affinity-purified polyclonal antibodies were: neurofilaments, AB1991 (Chemicon, Temecula, CA); MUNC18 (ABR); syntaxin (Sigma); SV2 (Stressgen, Vancouver, British Columbia, Canada); Ca V 2.1, CW65 ( Fletcher et al., 2001 ), Ca V 2.2, CW21 (also termed CW14; McEnery et al., 1997 ).

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: A Syntaxin 1, Gα o , and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization

doi: 10.1523/JNEUROSCI.0346-04.2004

Figure Lengend Snippet: Ab571 staining localized release sites at the chick ciliary ganglion calyx nerve terminal. A, Primary hippocampal neuron culture stained with Ab571 (green) and monoclonal anti-syntaxin (red). Note Ca channel puncta are along the same trajectories as the syntaxin staining, consistent with the synaptic contacts formed by a nerve fiber process onto an underlying postsynaptic neuron (data not shown). B, As for A except double-stained with monoclonal anti-SV2 (red). Some colocalization of the two dyes was noted in both A and B, but puncta of single stained syntaxin, SV2, or Ca channels were also evident. C, An optical section through a calyx presynaptic terminal attached to postsynaptic ciliary neuron stained for Ca channels (Ab571; green, top panel) and both Ab571 and synaptic vesicle clusters (SV2; red, bottom panel). A diagram (not to scale) of the synapse is presented in the bottom panel. D, Calyx terminal stained with Ab571 (top left panel) rabbit polyclonal SV2 (top center panel), and Na-K pump (top right panel) using the pretty-poly multiple polyclonal immunostaining technique (see Results). The next three (middle row) panels show colocalization of Ab517 and SV2 (left panel, middle row), Ab571 and Na-K pump (center panel, middle row), and SV2 and Na-K pump (right panel, middle row). The bottom panel is a triple overlay of the three stains. The ciliary neuron is above the calyx. Note the Ca channel clusters on the transmitter release face aspect and the secretory vesicle clouds overlapping the channels and distal from the release site. The staining in the image has been adjusted digitally to enhance the resolution of the three structures. Some distortion of sizes (such as surface membrane thickness) is a result of both the membrane coursing through the image plane at an angle and of limitations in the resolution of the light microscope. Scale bars: A, B, 5 μm; C,D, 2 μm.

Article Snippet: Affinity-purified polyclonal antibodies were: neurofilaments, AB1991 (Chemicon, Temecula, CA); MUNC18 (ABR); syntaxin (Sigma); SV2 (Stressgen, Vancouver, British Columbia, Canada); Ca V 2.1, CW65 ( Fletcher et al., 2001 ), Ca V 2.2, CW21 (also termed CW14; McEnery et al., 1997 ).

Techniques: Staining, Immunostaining, Light Microscopy

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Nicotinamide Adenine Dinucleotide-Dependent Binding of the Neuronal Ca 2+ Sensor Protein GCAP2 to Photoreceptor Synaptic Ribbons

doi: 10.1523/JNEUROSCI.3701-09.2010

Figure Lengend Snippet: Antibodies and labeling reagents

Article Snippet: More details on the antibodies used are given in . table ft1 table-wrap mode="anchored" t5 caption a7 Antibodies/reagents Source Dilution used Secondary antibody Dilution used Antibodies used for Western blotting GCAP2 6th immune serum Rabbit polyclonal 1:1000 GAR-POX; Sigma, cat. #A6154 1:10,000 GCAP2(A1); Santa Cruz Biotechnology, cat. #SC-59543 Mouse monoclonal 1:1000 GAM-POX; Sigma, cat. #A3673 1:10,000 U2656 ( Schmitz et al., 2000 ) Rabbit polyclonal 1:10,000 GAR-POX; Sigma, cat. #A6154 1:10,000 RIBEYE/CtBP2; BD Transduction Laboratories, cat. #612044 Mouse monoclonal 1:10,000 GAM-POX; Sigma, cat. #A3673 1:10,000 Antibodies used for immunolabeling GCAP2 6th immune serum Rabbit polyclonal 1:500 GAR-Cy3; Zymed, cat. #81-6115 1:1000 GCAP2(A1); Santa Cruz Biotechnology, cat. #SC-59543 Mouse monoclonal 1:1000 GAM-Cy3; Rockland, cat. #610-104-121 1:1000 U2656 ( Schmitz et al., 2000 ) Rabbit polyclonal 1:1000 GAR-Cy2; Rockland, cat. #611-111-122 1:1000 RIBEYE/CtBP2; BD Transduction Laboratories, cat. #612044 Mouse monoclonal 1:500 GAM-Cy2; Jackson ImmunoResearch, cat. #115-096-146 1:1000 mGluR6; Neuromics/Acris, cat. #RA13105 Rabbit polyclonal 1:500 GAR-Cy3; Sigma, cat. #C 2821 1:1000 SV2 A; Synaptic Systems, cat. #119 00 2 Rabbit polyclonal 1:500 GAR-Cy2; Rockland, cat. #611-111-122 1:1000 Synaptophysin; Sigma, cat. #S5768 Mouse monoclonal 1:500 GAM-Cy2; Jackson ImmunoResearch, cat. #115-096-146 1:1000 Antibodies used for whole-mount immunostaining U2656 ( Schmitz et al., 2000 ) Rabbit polyclonal 1:500 GAR-Cy3; Zymed, cat. #81-6115 1:1000 Reagent used for PNA labeling Lectin PNA from Arachis hypogaea a Lectin PNA; Invitrogen cat. #L-21409 1:250 Open in a separate window Antibodies used for in situ proximity ligation assays are summarized in detail in Materials and Methods. cat., Catalog; GAR, goat anti-rabbit; GAM, goat anti-mouse; PNA, peanut agglutinin. a Alexa Fluor 488 conjugate.

Techniques: Labeling, Western Blot, Transduction, Immunolabeling, Immunostaining