Adult DRG were dissected and collected in Hank’s Balanced Salt Solution (HBSS) on ice and digested according to standard procedures. Cultures were maintained in medium containing B27 and penicillin–streptomycin in DMEM:F12. Cells were plated on coated glass coverslips (0.01 mg ml−1PDL) for 48-72 h and fixed with 4% PFA–4% sucrose. Immunocytochemistry was performed by incubating fixed cells with Cdkl5 (Sigma HPA002847), βIII-tubulin (Abcam ab18207), CaMKIIα (Thermo-Fisher MA1-048), (Abcam ab81887), Parvalbumin (Abcam ab555), GFP (Thermo-Fisher A-6455), HA (Abcam ab18181) antibodies at 4 °C overnight. This was followed by incubation with AlexaFluor-conjugated goat secondary antibodies according to standard protocols (Invitrogen). All cells were counterstained with Hoechst (Molecular Probes).
Alexa fluor conjugated goat secondary antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor-conjugated goat secondary antibodies are fluorescent-labeled secondary antibodies derived from goats. They are designed to bind and detect primary antibodies raised in various species, enabling visualization and analysis of target proteins or other biomolecules in life science research applications.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst, by Thermo Fisher Scientific (1 mentions)
Parvalbumin, by Abcam (1 mentions)
βiii tubulin, by Abcam (1 mentions)
Cdkl5, by Merck Group (1 mentions)
Camkiiα, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Rabbit anti map2, by Abcam (1 mentions)
Vectamount aq, by Vector Laboratories (1 mentions)
Mouse anti syn1, by Synaptic Systems (1 mentions)
Chicken anti homer1, by Synaptic Systems (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
P pkc, by Cell Signaling Technology (1 mentions)
Mouse laminin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Balb c, by Charles River Laboratories (1 mentions)
C57bl 6 mice, by Charles River Laboratories (1 mentions)
Tunel assay kit, by Roche (1 mentions)
4 protocols using alexa fluor conjugated goat secondary antibodies
1
Immunocytochemical Analysis of DRG Neurons
2
Immunocytochemistry of Neuronal Synapses
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Neuron cultures (35–42 d after differentiation) on glass coverslips were fixed with 4% paraformaldehyde (PFA) and washed with PBS. After blocking and permeabilization with 5% normal goat serum (Vector), 1% BSA (Sigma-Aldrich) and 0.2% Triton- X-100 for 1 h at room temperature, cells were incubated with primary antibodies for mouse-anti-Syn1 (Synaptic Systems), chicken-anti-Homer-1 (Synaptic Systems), and rabbit-anti-MAP-2 (Abcam; Table 1 ) overnight at 4°C. After washing, secondary labeling was performed using appropriate Alexa Fluor-conjugated goat secondary antibodies (Invitrogen) for 2 h at room temperature. Coverslips were mounted using Vectamount AQ (H-5501; Vector Laboratories).
3
Neuronal Cell Characterization by Immunocytochemistry
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Glass coverslips were coated with 0.1 mg/ml Poly-D-Lysine (PDL), washed, and coated with mouse Laminin 2 µg/ml (Millipore). Cells were plated on coated coverslips for 24 h, at which time they were fixed with 4% PFA and then cryoprotected using 4% sucrose. Immunocytochemistry was performed by incubating fixed cells with anti-βIII Tubulin (1:1000, Tuj1, Mouse, Promega G7121), pSTAT3 (1:1000, Rabbit, Cell Signaling Technology 9145), and p-PKC (1:1000, Rabbit, Cell Signaling Technology 9371) antibodies at 4 °C O/N. This was followed by incubation with Alexa Fluor-conjugated goat secondary antibodies according to standard protocol (1:1000, Invitrogen). Slides were counterstained with DAPI to visualize nuclei (1:5000, Molecular Probes).
4
Isolation and Characterization of Transgenic Mice
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Balb/C and c57BL/6 mice were purchased from Vital River Laboratories (Beijing). GFP-transgenic mice (FVB.Cg-Tg(ACTB-EGFP)B5Nagy/J; Jackson Laboratory, Bar Harbor, ME, USA) were bred or maintained in the animal facility in Beijing Khasar Medical Technology. The mice received food and water ad libitum. The animal protocols were approved by the Beijing Khasar Medical Technology Animal Regulatory Office. All animal procedures followed the guidelines of the Animal Regulatory Office and relevant national and international guidelines. Octamer-binding transcription factor (Oct4), (sex-determining region Y)-box 2 (Sox2), E-cadherin, DNA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). An anti-GFP antibody was obtained from Aves Lab (Tigard, OR, USA). Alexa Fluor-conjugated goat secondary antibodies were from Invitrogen (Carlsbad, CA, USA). The TUNEL assay kit was from Roche Life Science (Shanghai). Other chemicals were from Sigma Chemical (Shanghai).
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