Primary hippocampal neurons were fixed with 4% paraformaldehyde for 20 minutes. After washing with Dulbecco's PBS (DPBS), the cells were permeabilized for 15 minutes with 0.1% PBST (DPBS containing 0.1% Triton X-100) and blocked for 30 minutes with 5% bovine serum albumin dissolved in DPBS. Cells were then incubated overnight at 4°C with primary antibody (NR2B, 1:200; or developmentally regulated brain protein [Drebrin], mouse, 1:200, Abcam, Cat# ab12350, RRID: AB_299034), and washed five times in DPBS. Secondary antibodies (goat anti-rabbit IgG-AlexaFluor 488, 1:200, Cell Signaling Technology, Danvers, MA, USA, Cat# 4412, RRID: AB_1904025; or goat anti-mouse IgG H&L [Cy3®, 1:200, Abcam, Cat# ab97035, RRID: AB_10680176]) were diluted with 5% bovine serum albumin and incubated at room temperature (25°C) for 1 hour. Subsequently, the cells were washed five times in DPBS. Images were acquired with a LAS X SP-5 confocal microscope (Leica DM IRB, Wetzlar, Germany). The colocalized NR2B/Drebrin area was quantified with ImageJ software.
Ab12350
Manufactured by Abcam
Sourced in Denmark
Ab12350 is a laboratory product developed by Abcam. It is a core piece of lab equipment with a specific function. No further details are available without extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg h l cy3, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Ab42824, by Abcam (1 mentions)
Dapi nuclear stain, by Thermo Fisher Scientific (1 mentions)
Acti stain 488 phalloidin, by Cytoskeleton (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Mab3420, by Merck Group (1 mentions)
A1 ti confocal microscope, by Nikon (1 mentions)
4 protocols using ab12350
1
Immunocytochemistry of Hippocampal Neurons
2
Immunofluorescence Labeling of Rat Hippocampal Neurons
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Rat hippocampal neurons grown on coverslips were fixed with 4% paraformaldehyde (PFA) at 37 °C for 10 min and then permeabilized with 0.5% triton-x 100 for 10 min. Nonspecific binding was blocked by incubation in PBS with 5% donkey serum, 2% goat serum and 1% BSA for 60 min at RT, followed by specific primary antibody incubation: mouse anti-Drebrin (Abcam, ab12350), rabbit anti-cofilin (abcam ab42824), mouse anti-α-tubulin (Abcam, ab7291) or mouse anti-Tau1 (Millipore, MAB3420) was added for incubation for 120 min at RT. The respective anti-mouse anti-rabbit Alexa fluor -488, -568 or -647 labeled secondary antibody was added for 60 min at room temperature. Primary and secondary antibodies were diluted in PBS with 2.5% donkey serum, 1% goat serum and 0.5% BSA. After primary as well as secondary antibody incubation three washing steps with PBS were performed. For F-actin labelling Acti-stain 488 phalloidin (Cytoskeleton) was used at 1 to 120 dilution in 1X PBS and incubated for 30 min at RT followed by 3 short PBS rinses. Coverslips were mounted onto the slides using ProLong Gold (Invitrogen) with or without nuclear stain DAPI (Invitrogen) and were stored light-protected.
3
Drebrin Binding Assay in Neurons
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For the binding assay, cells were incubated at in vitro day 14 (DIV14) for 48 hours with either commercial anti-Drebrin antibody (ab12350, Abcam; 20μg/ml, 10μg/ml, 2μg/ml, 1μg/ml) or corresponding concentrations of human control IgG (Intratect; Orifarm, Odense, Denmark). Cells were washed with PBS and immediately fixed with PFA in PBS at DIV16 for 10 minutes, followed by 3 washing steps. Neurons were permeabilized with PBS/Triton X-100 (0.3% [wt/vol]) for 10 minutes, blocked in buffer containing PBS/Triton X-100 (0.1% [wt/vol]) with 1% BSA and 10% NGS for 1 hour at RT, and incubated overnight with Alexa Fluor secondary antibodies (goat antihuman A11013, Invitrogen; goat antimouse A11001, Invitrogen; 1:1,000) and DAPI (1:100). The next day, cells were washed 3 times and mounted with Mowiol. Images were taken with a laserscanning Nikon A1/Ti confocal microscope.
4
Quantifying Synapse Morphology from Patient IgG
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For functional analyses, IgG fraction from Patient 1 serum was purified via Protein G HP Ab Spin Trap Kit (GE Healthcare, Chicago, IL; 28-4083-47) according to the manufacturer's protocol. PHNs on coverslips were incubated at DIV14 for 20 minutes or 48 hours either with affinity-purified anti-Drebrin patient IgG fraction or with control IgG. After incubation, cells were washed, fixed, permeabilized, and blocked as described above. After incubation with primary (anti-Drebrin, ab12350, Abcam, 1:1,000; anti-Homer, 160004, Synaptic Systems, 1:1,000) and appropriate secondary antibodies, cells were washed and mounted. Images were taken with a laser-scanning Nikon A1/Ti confocal microscope.
For each condition, 3 biological and 6 technical replicates captured in 5 stacks (0.3μm steps) were analyzed. Synapses were identified by using Homer1 staining and analyzed by generating the maximum intensity projection (ImageJ). Intensity was set as a 20% threshold from the maximum intensity of the Homer1 staining. Synapse size was set between 0.02 and 0.4 μm 2 .
For each condition, 3 biological and 6 technical replicates captured in 5 stacks (0.3μm steps) were analyzed. Synapses were identified by using Homer1 staining and analyzed by generating the maximum intensity projection (ImageJ). Intensity was set as a 20% threshold from the maximum intensity of the Homer1 staining. Synapse size was set between 0.02 and 0.4 μm 2 .
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