For immunohistochemical analysis, rabbit cornea, kidney, muscle, and optic nerve were embedded in an optimum cutting temperature compound and processed into 5 μm frozen sections. Tissue sections were treated with three kinds of antibodies, IV-3A9 (Daiichi Fine Chemical, Toyama, Japan), JK132, and #370. Secondary antibody was horseradish peroxidase (HRP) conjugated anti-mouse IgG (Jackson immune Research Laboratories, West Grove, PA). The sections were stained by the treatment with 3,3′-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. For double immunofluorescence staining, frozen sections were prepared into 25 μm thickness and whole mount rabbit corneal tissues were fixed with 70% ethanol. These specimens were stained with mouse monoclonal antibodies, anti-CD31 (Dako, Glostrup, Denmark) or IV-3A9 were detected by the treatment with Alexa 594 conjugated anti-mouse IgG (Jackson Immuno Research Laboratories), biotin conjugated #370 was detected by the treatment with Alexa 488 conjugated streptavidin (Jackson Immuno Research Laboratories), and observed with a confocal laser scanning microscope (LSM-510) (Carl Zeiss, Oberkochen, Germany).
Alexa 488 conjugated streptavidin
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor 488 conjugated streptavidin is a fluorescent labeling reagent used for the detection and visualization of biotinylated biomolecules. Streptavidin, a bacterial protein, binds tightly to biotin, allowing for the sensitive detection of biotinylated targets. The Alexa Fluor 488 dye provides a bright green fluorescent signal upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (2 mentions)
Anti cd31, by Agilent Technologies (1 mentions)
Mouse anti beta actin antibody, by Merck Group (1 mentions)
Streptavidin hrp, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
3h thymidine, by PerkinElmer (1 mentions)
Anti phospho p38 mapk, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 goat anti rabbit, by Cell Signaling Technology (1 mentions)
Sp8 laser scanning confocal microscope, by Leica (1 mentions)
Las af software, by Leica (1 mentions)
5 protocols using alexa 488 conjugated streptavidin
1
Immunohistochemical Analysis of Rabbit Tissues
2
Antibody and Cell Culture Reagents
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Biotin-conjugated goat anti-HA IgG was from Vector laboratories (Burlingame, CA). Streptavidin-HRP, Alexa-488 conjugated streptavidin, and rhodamine-conjugated streptavidin were from Jackson ImmunoResearch Laboratories (West Grove, PA). DMEM, mouse anti-beta-actin antibody, sodium bicarbonate and p-nitrophenyl acetate (NPA) were from Sigma-Aldrich (St. Louis, MO). 4',6-diamidino-2-phenylindole (DAPI) and RPMI-1640 were from Invitrogen (Carlsbad, CA). Bovine calf serum (BCS) was from HyClone (Logan, Utah). 3H-thymidine was from PerkinElmer (Boston, MA).
3
Immunofluorescence Imaging of Phosphorylated p38 MAPK and PKCα
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The immunofluorescence technique (including the perfusion method) was performed as previously described (Almeida-Pereira et al. 2016) (link). In brief, after sections were blocked in PBS buffer containing 10% normal horse serum, they were incubated overnight with primary antibodies rabbit anti-phospho-p38 MAPK (1:1000; #9211, Cell Signaling Technology) or anti-PKCα (1:500; #2056, Cell Signaling Technology) and guinea pig anti-AVP (1:40,000; T-5048, Peninsula Laboratories, San Carlos, CA, USA) or anti-OT (1:40,000; T-5021, Peninsula Laboratories) at 4°C. After washing, the sections were incubated with secondary antibody donkey anti-guinea pig CY5 (1:200; Jackson ImmunoResearch Laboratories), biotinylated secondary antibody donkey anti-rabbit (1:200; Vector Laboratories Inc., Burlingame, CA, USA) and secondary antibody Alexa 488-conjugated streptavidin (1:200; Jackson ImmunoResearch Laboratories) for PKCα immunofluorescence and incubated with secondary antibody goat anti-rabbit Alexa Fluor 488 (1:200; Cell Signaling Technology) for phospho-p38 MAPK at room temperature. Images were collected on a Leica TCS SP5 confocal microscope system equipped with 488 nm (argon-krypton) and 633 nm (helium-neon) laser lines (#2004/08868-0, Sao Paulo Research Foundation-FAPESP). For each nucleus, all images were detected at identical acquisition settings.
4
Whole-cell patch-clamp recordings of goldfish cone photoreceptors
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Whole-cell patch-clamp recordings (current-clamp configuration with I = 0) from the inner segments of individual cones in intact goldfish neural retinas were made under visual control, as described previously (Ribelayga et al., 2008 (link)). The pipette solution contained (in mM) 20 KCl, 100 D-K-gluconate, 7.48 KHCO3, 5.0 HEPES, 1.0 MgCl2, 4.0 Na2-ATP, 0.02 Na3-GTP, 1.0 CaCl2, and 1.0 EGTA. The pH was adjusted to 7.3 with KOH and the solution was divided into aliquots and kept frozen. Neurobiotin (0.3%) was added fresh daily to a sample of pipette solution. Individual cones were labeled by iontophoresis of the biotinylated Neurobiotin tracer (MW = 373) during whole-cell patch-clamp recording by maintaining the recorded cone at +20 mV for 10 min. After an additional 30 min in the dark, retinas were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PH 7.4) at room temperature for 1 h. The tissues then were washed and labeled. Fluorescent photoreceptors, indicating the presence of Neurobiotin tracer, were subsequently visualized with streptavidin-conjugated-Alexa 488 (Jackson ImmunoResearch, West Grove, PA, USA). Labeled cells were imaged, photographed, and counted with a Leica SP8 laser scanning confocal microscope (Leica Microsystems Inc, Buffalo Grove, IL, USA). Tracer-coupled rods and cones were counted with NIH ImageJ software (Ribelayga et al., 2008 (link)).
5
Visualizing GFP-labeled Hippocampus Neurons
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Mice were transcardially perfused with cold 4% paraformaldehyde in PBS for 20 min and processed for histology.56 (link) Brains were postfixed in the same solution overnight at 4°C and stored in 30% (w/v) sucrose at 4°C. Serial sections (25 μm) were cut coronally through the entire hippocampus and stored in 50% glycerol in PBS at 20°C. To detect green fluorescent protein (GFP), sections were incubated overnight with biotinylated goat anti-GFP antibody (a11122, Thermo Fisher) at pH 7.4, 4°C, containing 0.3% Triton X-100 in PBS (PBST). The next day, the sections were washed three times with 1% NGS and 0.3% Triton X-100 in PBS for 15 min and incubated with streptavidin-conjugated Alexa488 (code 43016480084, Jackson Immunoresearch) secondary antibodies in 1% normal goat serum in PBS for 2 h. Optical sectioning was used during the acquisition of Z-stack images. LAS AF software (Leica Microsystems, Wetzlar, Germany) was used to sharpen images, adjust brightness and contrast levels, and compose Z-projection (maximum intensity) and channel-merged figures. For dendritic analysis, the samples were photographed in the molecular layer of DG.
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