Animals were euthanized with Isoflurane, decapitated, and enucleated. Retinas were dissected in cold oxygenated mouse artificial cerebrospinal fluid (mACSF, pH 7.4) containing (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1 NaH2PO4, 11 glucose, and 20 HEPES. During retina dissections note was taken of retinal location and orientation (Wei et al., 2010 (link)). Whole-mount immunolabelings were performed on retinas that were flattened on a filter paper (Millipore, HABP013). Flat-mounted retinas were fixed for 15 mins in 4% paraformaldehyde (in mACSF). After rinses in phosphate buffer (PBS), the retinas were pre-incubated in blocking solution containing 5% donkey serum and 0.5% Triton X-100 (in PBS) followed by incubation with primary antibodies in blocking solution over 3–4 nights at 4°C. Primary antibodies used were directed against: GFP (1:1000, chicken, Abcam), GABAAα3 (1:3000, guinea pig, J.M. Fritschy), GABAAα1 (1:5000, guinea pig, J.M. Fritschy), GlyRα1 (1:500, mouse monoclonal mAb2b, Synaptic Systems) and gephyrin (1:1000, mouse monoclonal clone 3B11, Synaptic systems). Secondary antibody incubation was carried out overnight in PBS using anti-isotypic Alexa Fluor (1:1000, Invitrogen) or DyLight conjugates (1:1000, Jackson Immunoresearch). Retinas were subsequently mounted on slides using Vectashield mounting medium (Vector Labs).
Mab2b
Manufactured by Synaptic Systems
Sourced in Germany
MAb2b is a monoclonal antibody that can be used as a laboratory tool. It is designed to detect and bind to a specific target molecule. The core function of MAb2b is to serve as a detection and identification reagent in various experimental and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Habp013, by Merck Group (2 mentions)
Dylight conjugates, by Jackson ImmunoResearch (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Mab7a, by Synaptic Systems (1 mentions)
Ix81 inverted fluorescence microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Pdsred monomer mem, by Takara Bio (1 mentions)
Mowiol 4 88, by Carl Roth (1 mentions)
Jung frigocut 2800e, by Leica (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Alexa fluor 488, by Dianova (1 mentions)
5 protocols using mab2b
1
Whole-Mount Immunolabeling of Retinal Neurons
2
Quantitative Visualization of Glycine Receptors
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Fixed cells were labelled with primary antibodies (rabbit anti-GlyRα1, custom made; mouse anti-gephyrin mAb7a, Synaptic Systems 147011) and secondary antibodies (Cy3-conjugated goat anti rabbit IgG and Alexa Fluor 647 goat anti mouse). For PALM counting of receptor-associated mEos2-gephyrin (Fig. 2F ), mouse spinal cord neurons were fixed and endogenous GlyRs were labelled with QDs emitting at 605 nm (primary antibody: mouse anti-GlyRα1, Synaptic Systems mAb2b; secondary antibody: anti-mouse Fab’-QD605).
3
Immunostaining of GlyR Receptors
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To stain GlyR surface receptors, GlyRα1 variants, and pDsRed-Monomer-Mem [Takara Bio (formerly Clontech), Mountain View, CA, United States] were co-transfected into HEK293 cells. All steps were performed at room temperature. After fixation for 20 min with 50 μl 4% paraformaldehyde, 4% sucrose solution, cells were washed three times with PBS and blocked for 30 min with 5% (v/v) goat serum in PBS. Afterward, cells were incubated for 1 h with the GlyRα1-specific primary antibody mAb2b (1:500 in blocking solution; epitope amino acids 1–10 of mature GlyRα1; Synaptic Systems, Göttingen, Germany). Cells were washed three times with PBS and incubated for 45 min with the secondary Alexa488-coupled goat-anti-mouse antibody (1:500 in blocking solution; Dianova, Hamburg, Germany). Then, cells were washed three times with PBS, incubated for 5 min with DAPI (1:5000 in PBS; Thermo Fisher Scientific, Waltham, MA, United States) and mounted on glass slides with Mowiol 4-88 (Carl Roth, Karlsruhe, Germany). Imaging was performed using an Olympus IX-81 inverted fluorescence microscope (Olympus, Tokyo, Japan).
4
Immunostaining of Spinal Cord Neurons
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Spinal cords were dissected, rapidly frozen on dry ice, and embedded in Tissue-Tek (Sakura Finetek). Transverse slices with a thickness of 8–9 μm were cut with a cryostat (Jung Frigocut 2800E, Leica). Four to six slices were transferred to glass slides (25 × 75 × 1.0 mm; SuperFrostR Plus, Menzel Gläser/Thermo Scientific) and stored for further analysis at −80°C. For immunostaining, tissue slides were fixed for 30 s in ice-cold 2% PFA and immersed once in 50 mm NH4Cl following quenching for 30 min in 0.1 mm glycine in PBS. Tissue slices were blocked by 10% normal horse serum in PBS for 1 h at room temperature. The slides were incubated with antibodies GlyR α1 (1:250; mAb2b; catalog #146 111, Synaptic Systems), VGAT (1:300; catalog #131 003, Synaptic Systems), gephyrin (1:200; catalog #147 003, Synaptic Systems), and synapsin (1:300; catalog #574778, Calbiochem) in 10% normal horse serum in PBS overnight at 4°C. After three washing steps with PBS for 10 min each, tissue was incubated with secondary antibodies coupled to Cy3 and Alexa Fluor 488 (1:1000, Dianova) diluted in 10% normal horse serum in PBS for 45 min at 22°C. For staining of the cell nuclei, slides were incubated in Molecular Probes DAPI solution (Thermo Fisher Scientific) diluted 1:1500 in PBS for 10 min at room temperature in a dark chamber. Finally, the slides were mounted with aqua polymount (Polysciences).
5
Whole-Mount Immunolabeling of Retinal Neurons
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Animals were euthanized with Isoflurane, decapitated, and enucleated. Retinas were dissected in cold oxygenated mouse artificial cerebrospinal fluid (mACSF, pH 7.4) containing (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1 NaH2PO4, 11 glucose, and 20 HEPES. During retina dissections note was taken of retinal location and orientation (Wei et al., 2010 (link)). Whole-mount immunolabelings were performed on retinas that were flattened on a filter paper (Millipore, HABP013). Flat-mounted retinas were fixed for 15 mins in 4% paraformaldehyde (in mACSF). After rinses in phosphate buffer (PBS), the retinas were pre-incubated in blocking solution containing 5% donkey serum and 0.5% Triton X-100 (in PBS) followed by incubation with primary antibodies in blocking solution over 3–4 nights at 4°C. Primary antibodies used were directed against: GFP (1:1000, chicken, Abcam), GABAAα3 (1:3000, guinea pig, J.M. Fritschy), GABAAα1 (1:5000, guinea pig, J.M. Fritschy), GlyRα1 (1:500, mouse monoclonal mAb2b, Synaptic Systems) and gephyrin (1:1000, mouse monoclonal clone 3B11, Synaptic systems). Secondary antibody incubation was carried out overnight in PBS using anti-isotypic Alexa Fluor (1:1000, Invitrogen) or DyLight conjugates (1:1000, Jackson Immunoresearch). Retinas were subsequently mounted on slides using Vectashield mounting medium (Vector Labs).
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