The following commercial antibodies were used for immunofluorescent imaging and immunoblotting: Cux1 (Santa Cruz Biotechnology, sc13024), Neurofilament (Covance, SMI-32R), βTubulin III (Sigma, T2200), Flag clone M2 (Sigma, F1804), MAP2 clone HM2 (Sigma, M4403), Tbr2 (Millipore, AB2283), Kif1a (BD Bioscience, 612094), and EGFP (AVES, GFP-1010). All other antibodies were custom-made in the Südhof laboratory.
Map2 clone hm 2
Manufactured by Merck Group
Sourced in United States
MAP2; clone HM-2 is a laboratory product manufactured by Merck Group. It is a protein that serves as a core function in research and development applications. Detailed specifications and intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain elite abc, by Vector Laboratories (2 mentions)
Biotinylated secondary antibody, by Vector Laboratories (2 mentions)
Flag clone m2, by Merck Group (1 mentions)
β tubulin 3, by Merck Group (1 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
Nickel ammonium sulfate, by Merck Group (1 mentions)
β d glucose, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Ammonium chloride, by Merck Group (1 mentions)
Alexa fluor 488 donkey anti rabbit igg h l antibody, by Thermo Fisher Scientific (1 mentions)
Prolong antifade reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (1 mentions)
Ferritin, by Merck Group (1 mentions)
Nf clone nf 01, by Exbio (1 mentions)
Secondary fl uorescein and rhodamineconjugated antibodies, by Merck Group (1 mentions)
Vimentin clone v9, by Merck Group (1 mentions)
7 protocols using map2 clone hm 2
1
Immunofluorescent Imaging and Immunoblotting Protocols
2
Identifying Cortical Neuron Subtypes
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The cell types of micropatterned cortical neurons were retrospectively identified by immunofluorescence staining. The cultured neurons were fixed using the method described previously [6 (link)]. The following antibodies were used: anti-microtubule associated protein 2 (MAP2) clone HM-2, a mouse monoclonal IgG1 antibody from Sigma diluted 1:1000 to 1:2,000; anti-GAD67 clone 1G10.2, a mouse monoclonal IgG2a antibody from Chemicon diluted 1:1,000; anti-tau-1 clone PC1C6, a mouse monoclonal IgG2a antibody from Chemicon diluted 1:350; Alexa 488-labelled anti-mouse IgG1 antibody from Molecular Probes diluted 1:2,000; Alexa 594-labelled anti-mouse IgG2a antibody from Molecular Probes diluted 1:1,000. The antibodies for MAP2 and tau-1 stain the somatodendritic domain and the axonal process, respectively, of both excitatory and inhibitory neurons. While there is no effective excitatory neuron marker that is available for use in the early stages of culture, GAD67 is a reliable marker for GABAergic neurons, which are major inhibitory cells in the cortex (S2 Fig ) [12 (link),15 (link),16 (link)].
3
Immunohistochemistry Protocol for Neuronal and Glial Markers
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Immunohistochemistry was performed as previously described [31 (link)]. In brief, sections were de-paraffinized with a graded series of xylene and ethanol. Antigen retrieval was done by boiling the sections in citrate buffer (0.01 M, pH 6) for 10 min. Sections were thereafter incubated with H2O2 (3%) for 10 min for blocking of peroxidases and blocked with normal horse serum (Vector Laboratories, Burlingame, CA, U.S.A.) for 30 min. Sections were incubated with primary antibodies against microtubule-associated protein 2 (MAP2; clone HM-2, 1:1000; Sigma-Aldrich, St. Louis, MO, U.S.A.) or ionized calcium-binding adapter molecule 1 (Iba-1; Cat no 019-19741, Fujifilm Wako Chemicals U.S.A. Corporation, Richmond, VA, U.S.A.) overnight at 4°C. The sections were then incubated with the corresponding biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, U.S.A.) for 60 min at room temperature. Peroxidase staining was detected using Vectastain ABC Elite (Vector Laboratories, Burlingame, CA, U.S.A.) with 3,3-diaminobenzidine (0.5 mg/ml) enhanced with ammonium nickel sulfate (15 mg/ml), β-D-glucose (2 mg/ml), ammonium chloride (0.4 mg/ml), and β -glucose oxidase (0.01 mg/ml; all chemicals purchased from Sigma-Aldrich, St. Louis, MO, U.S.A.). Sections were dehydrated with graded series of ethanol and xylene and mounted using Pertex Histofix (Histolab, Askim, Sweden).
4
Brain Tissue Histopathological Analysis
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Resected brain specimens were formalin-fixed overnight in 4% formalin and processed into liquid paraffin according to standardized histopathology protocols [5 (link)]. All sections were cut at 4 μm, mounted on positively charged glass slides (Superfrost, Germany) and stained with hematoxylin and eosin (H&E) or Nissl-Luxol-Fast-Blue (Nissl-LFB). Immunohistochemical stainings were performed on selected slides followed by hematoxylin counterstaining. The following antibodies were used according to manufacturer’s protocols: NeuN (Clone A60; Millipore, Temecula, USA), MAP2 (clone HM-2, Sigma, St. Louis, USA), Mib1 (clone Ki67, cell marque, Rocklin, USA), Olig2 (clone JP18953, IBL International, Hamburg, Germany), and CNPase (clone 11-5B, Millipore).
5
Multicolor Immunocytochemistry of Neuronal Structures
6
Immunohistochemical Staining Protocol
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Sections were de-paraffinized in a graded series of xylene followed by re-hydration in decreasing concentrations of ethanol and boiled in citrate buffer (pH 6) for 10 min for antigen retrieval. Peroxidases were blocked by H2O2 (3%) treatment for 10 min. To block unspecific binding, sections were treated with 3% horse serum (for MAP2 staining) or 4% goat serum with 0.1% Triton-X100 (for Iba-1 staining) for 30 min. Sections were then incubated overnight at 4 °C with primary antibody: microtubulin-associated protein 2 (MAP2; clone HM-2, 1:1000; Sigma-Aldrich, St. Louis, MS, USA) or ionized calcium-binding adapter molecule 1 (Iba-1; 1:2000, Cat# 019-19741; Fujifilm Wako Chemicals U.S.A. Corporation, Richmond, VA, USA), and then incubated in corresponding biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for 60 min at room temperature.
After incubating sections with Vectastain ABC Elite (Vector Laboratories, Burlingame, CA, USA), they were developed with 0.5 mg/mL of 3,3-diaminobenzidine enhanced with 15 mg/mL ammonium nickel sulfate, 2 mg/mL β-D-glucose, 0.4 mg/mL ammonium chloride and 0.01 mg/mL β -glucose oxidase. Sections were then de-hydrated in a graded series of ethanol and xylene and mounted using Pertex mounting medium (Histolab, Askim Sweden). All chemicals were purchased from Sigma-Aldrich (St. Louis, MS, USA) unless otherwise stated.
After incubating sections with Vectastain ABC Elite (Vector Laboratories, Burlingame, CA, USA), they were developed with 0.5 mg/mL of 3,3-diaminobenzidine enhanced with 15 mg/mL ammonium nickel sulfate, 2 mg/mL β-D-glucose, 0.4 mg/mL ammonium chloride and 0.01 mg/mL β -glucose oxidase. Sections were then de-hydrated in a graded series of ethanol and xylene and mounted using Pertex mounting medium (Histolab, Askim Sweden). All chemicals were purchased from Sigma-Aldrich (St. Louis, MS, USA) unless otherwise stated.
7
Identifying Brain Cell Types
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To identify astroglia cell types, we used antibodies against GFAP (clone GF-01, 1:100, Exbio, Prague), and polyclonal sera to GFAP (1:100, Dako). Oligodendroglia cells were detected by antibodies to GalC (clone mGalC, 1:10, Boehringer-Mannheim, Vienna) and to O4 antigen (clone 81, 1:10, Boehringer-Mannheim, Vienna). Microglial cells were defi ned as immunoreactive with antibodies to CD11c (clone BU15, 1:50, Immunotech, France), and polyclonal sera to ferritin (1:50, Sigma). Neuronal cells were identifi ed with antibodies to MAP2 (clone HM-2, 1:50, Sigma), and NF (clone NF-01, 1:100, Exbio, Prague). The endothelial cells were detected by polyclonal sera against Von Willebrand Factor-VIII-related antigen (1:100, Dako). The antibodies to vimentin (clone V9, 1:100, Sigma), to cytokeratins monoclonal anti-pan CK (types: 1,4,5,6,8,10,13,18,19, 1:100, Sigma), and polyclonal sera against fi bronectin (1:100, Sigma) were used for further characterization of human brain cells. The secondary fl uorescein-and rhodamineconjugated antibodies were purchased from Sigma and Sevapharma (Prague).
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