The tissue was processed for array tomography as described in Micheva and Smith (20 (link)). Visual cortex was dissected from perfused animals and embedded in LR white resin using a bench top protocol. Ribbons with between 70–100 serial 70 nm ultrathin sections were prepared from P30 Mecp2-KO, WT and Mecp2-KO/NR2A-Het mice using an ultramicrotome (UC7, Leica Microsystems) and mounted side by side on subbed glass coverslips. Coverslips were immunostained using synapsin I (mouse, SYSY), synaptophysin (mouse, Abcam), PSD-95 (rabbit, Cell Signaling Technology), GluR1 (rabbit, Millipore), GluR2/3 (rabbit, Abcam), GluN2A or GluN2B (rabbit, Frontier Science Co., Ltd) and parvalbumin (guinea pig, Frontier Science Co., Ltd) antibodies. For secondary antibodies, Alexa 488, Alexa 594 and Alexa 647 (Invitrogen) were used from the appropriate species. For some ribbons, applied antibodies were eluted and the sections were restrained with different antibodies. The identification and quantification of GluN2A- and 2B-positive synapses is described in Figure 2 (A–D) and in further detail in the Supplemental Methods .
Synapsin 1
Manufactured by Abcam
Sourced in United Kingdom, United States
Synapsin-1 is a neuronal phosphoprotein that plays a crucial role in the regulation of neurotransmitter release and synaptic vesicle trafficking. It is a key component of the presynaptic machinery and is involved in the anchoring and clustering of synaptic vesicles at the active zones of synapses.
Automatically generated - may contain errors
Lab products found in correlation
Psd95, by Abcam (5 mentions)
Neuronal nuclei (neun), by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Gabaarβ3, by Abcam (2 mentions)
Nf κb p65, by Abcam (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Proteintech (2 mentions)
Tnf α, by Abcam (2 mentions)
Il 10, by Abcam (2 mentions)
Il 1β, by Abcam (2 mentions)
Interleukin 6 (il 6), by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (2 mentions)
Traf6, by Abcam (2 mentions)
Synaptophysin, by Abcam (2 mentions)
Psd95, by Cell Signaling Technology (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
P synapsin, by Arigo Biolaboratories (2 mentions)
19 protocols using synapsin 1
1
Array Tomography for Synaptic Quantification
2
Immunofluorescence Analysis of Synaptic Markers in Cerebral Cortex
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Four-micron frozen cerebral cortex sections or PCAs cultured on glass coverslips were fixed with 4% paraformaldehyde for 30 min and then treated with 0.1% Triton X-100 for 10 min at room temperature. Blocking was achieved with PBS containing 5% normal goat serum for 1 h at room temperature. Sections were then incubated overnight at 4 °C with the following primary antibodies: GABAARβ1, GABAARβ3, TrkB, pAKT, synapsin I, PSD95, GAP43, VGAT, bassoon, and MAP2 (Abcam). Binding of primary antibodies was detected by incubating the sections for 30 min with FITC (green)/Alexa Fluor 594 (red)-conjugated secondary antibody. Imaging was performed with a Leica TCS SP2 confocal laser scanning microscope. For the analysis of synaptogenesis in PHNs, the neurons were co-stained with anti-VGAT and anti-bassoon antibodies. The number and size of the synapses were analyzed with the ImageJ software.
3
Synaptosome Purification and Protein Analysis
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Synaptosome purification has been described previously26 (link). For the validation of NBEA expression after retrieval, whole hippocampal tissue was used. For the rest of the study, the dorsal hippocampus was dissected and homogenized in ice-cold lysis buffer using a Teflon-glass tube. For immunoblotting analysis, antibodies targeting the following proteins were used. Primary antibodies against the following antigens were used: synapsin I (1:1,000, Abcam, Cambridge, UK), postsynaptic density 95 (PSD95; 1:1,000, Abcam, Cambridge, UK), histone H1 (1:1,000, Abcam, Cambridge, UK), NBEA (K-20) (1:1,000, Santa Cruz Biotech., Dallas, TX, USA), A kinase anchor protein 150 (AKAP150; 1:1,000, Santa Cruz Biotech, Dallas, TX, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000, Cell signaling, Danvers, MA, USA). The secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse (1:5,000) or anti-rabbit (1:10,000) antibodies (PerkinElmer Life Sciences, Norwalk, CT, USA). Signal detection was performed using the LAS 2000 system (LAS-2000, Fuji, Tokyo, Japan) with enhanced chemiluminescence (Western-CDP star, PerkinElmer Life Sciences, Norwalk, CT, USA). Densitometric analysis of immunoreactivity for each protein was conducted using NIH ImageJ software.
4
Primary Hippocampal Neuron Culturing Protocol
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The primary cultures of hippocampal neurons were prepared from day 18 embryonic Wistar rats (Charles River Laboratories Japan, Tokyo, Japan). Extracted hippocampi were dissociated using a dissociation kit (Sumitomo Bakelite, Tokyo, Japan). Isolated hippocampal neurons were plated on glass bottom dishes (Iwaki, Tokyo, Japan) coated with poly-D-lysine (PDL; Sigma-Aldrich, St. Louis, MO, USA) for fluorescence imaging or in 96-well plates for MTT assay, and cultured in neurobasal medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with B-27 (Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin (Nacalai Tesuque, Kyoto, Japan). The neurons were cultured at 37 °C in a humidified atmosphere of 5% CO2 for 7–9 days. Neuronal culture and synaptic formation among neurons were confirmed by immunofluorescence imaging of a neuron marker, βIII-tubulin (Sigma-Aldrich, St. Louis, MO, USA), and a synapse marker, synapsin I (Abcam, Cambridge, UK) (Figure S1 ).
5
Western Blot Analysis of Neuronal Proteins
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The total amount of protein in the lysates was determined by BCA protein assay (AMRESCO). Samples (50 μg protein) were separated by 10% SDS-PAGE and electroblotted to PVDF membranes, which were blocked by incubation in 5% non-fat dry milk dissolved in TBS-T (150 mM NaCl, 50 mM Tris, 0.05% Tween 20). Following transfer, proteins were probed using the following primary antibodies: D1R, DARPP32, GABAARβ1, GABAARβ3, TrkB, pAKT, AKT, synaptotagmin, synapsin I, PSD95, spinophilin, and β-actin (Abcam). Then, horseradish peroxidase-conjugated secondary antibody was used. After extensive washing, protein bands detected by antibodies were visualized by ECL reagent (Thermo) after exposure on Kodak BioMax film (Kodak).
6
Immunohistochemistry of Brain Tissues and Cultured Cells
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For brain tissues: four‐micron frozen cortical sections fixed in acetone or 4% formaldehyde. For cells cultured on glass coverslips were fixed with 4% paraformaldehyde for 30 minutes and then treated with 0.1% Triton X‐100 for 20 minutes at room temperature. Blocking was achieved with PBS containing 5% normal goat serum for 1 hour at room temperature. Sections were then incubated overnight at 4°C with the following primary antibodies: total‐Akt and pAkt‐Ser473 (Abcam), PSD95 (Abcam), synapsin I (Abcam), microtubule‐associated protein 2 (Map2) (Abcam), Beclin1 (Abcam); LC3B (Abcam). Binding of primary antibodies was detected by incubating the sections for 30 minutes with Alexa Fluor 488 (green)/Alexa Fluor 594 (red) conjugated secondary antibody (Abcam), or with horseradish peroxidase (HRP) ‐labelled secondary antibody (Abcam) and were visualized by diaminobenzidine (DAB).
7
Hippocampal Protein Analysis in Rats
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The hippocampal tissues were stripped individually from six rats in each group and lysed with radio immunoprecipitation assay buffer (RIPA, Beyotime Biotech, Shanghai, China) containing Phenylmethanesulfonyl uoride (PMSF, Beyotime, China). The extracted proteins were sonicated and incubated overnight at 4 °C and then centrifuged at 12000 rpm, 4 °C for 30 min. The supernatants were collected and the concentration of protein was determined by the Bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, China); Equal amounts of samples (30μg) was added to the wells and separated by SDS-PAGE gel and then the proteins were transferred onto polyvinylidene uoride (PVDF) membrane (Millipores, 0.45 μm, Billerica, MA, USA). After blocking with with 5% BSA at 37 °C for 1 h, the membranes were incubated with the following primary antibodies: TRAF6, IL-1β, IL-10, IL-6, TNF-α, iNOS, PSD95, Synapsin I, and NF-κB p65 (all diluted to 1:1000 and purchased from Abcam) and GAPDH (1:10000, Proteintech Biotech, Wuhan, China) at 4 °C overnight. The membranes were then incubated by the horseradish peroxidase-conjugated secondary antibody (1:10000, Proteintech) at 37 °C for 1 h. Enhanced chemiluminescence (ECL; Uscn Life, China) was used to detect the protein bands on a Chemi-Doc XRS + imaging system (Bio-Rad, USA). The gray value of all proteins was quanti ed by ImageJ software (NIH).
8
Hippocampal Protein Analysis in Rats
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The hippocampal tissues were stripped individually from six rats in each group and lysed with radio immunoprecipitation assay buffer (RIPA, Beyotime Biotech, Shanghai, China) containing Phenylmethanesulfonyl uoride (PMSF, Beyotime, China). The extracted proteins were sonicated and incubated overnight at 4 °C and then centrifuged at 12000 rpm, 4 °C for 30 min. The supernatants were collected and the concentration of protein was determined by the Bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, China); Equal amounts of samples (30μg) was added to the wells and separated by SDS-PAGE gel and then the proteins were transferred onto polyvinylidene uoride (PVDF) membrane (Millipores, 0.45 μm, Billerica, MA, USA). After blocking with with 5% BSA at 37 °C for 1 h, the membranes were incubated with the following primary antibodies: TRAF6, IL-1β, IL-10, IL-6, TNF-α, iNOS, PSD95, Synapsin I, and NF-κB p65 (all diluted to 1:1000 and purchased from Abcam) and GAPDH (1:10000, Proteintech Biotech, Wuhan, China) at 4 °C overnight. The membranes were then incubated by the horseradish peroxidase-conjugated secondary antibody (1:10000, Proteintech) at 37 °C for 1 h. Enhanced chemiluminescence (ECL; Uscn Life, China) was used to detect the protein bands on a Chemi-Doc XRS + imaging system (Bio-Rad, USA). The gray value of all proteins was quanti ed by ImageJ software (NIH).
9
Western Blot Analysis of Brain Proteins
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Example 4
Frozen brain tissues were homogenized in cold RIPA buffer with protease inhibitors and proteins were isolated and measured using BCA assay (BioRad, Mississauga, ON, Canada). Proteins were loaded at 50 μg per lane on a 12% polyacrylamide gel for CTR, RAMP3, NeuN, synapsin1, phospho-synapsin, and Iba1, or 4-20% polyacrylamide gradient gel (BioRad) for APP. Proteins were transferred to nitrocellulose membrane, and blocked with LiCOR blocking buffer. Blots were incubated with primary antibodies overnight at 4° C. on a shaker. Primary antibody used for CTR (1:1000 rabbit; Thermo Scientific), RAMP3 (1:1000 rabbit; Santa Cruz Biotechnology), 6E10 for APP (1:5000 mouse; 6E10; Covance), NeuN (1:1000, rabbit, Abcam), synapsin-1 (1:1000 rabbit, Abcam), synaptophysin (1:1000, rabbit, Abcam), phosphosynapsin (1:1000, rabbit, Abcam), Iba1 (1:1000, rabbit, Wako), β-tubulin (1:1000, rabbit, Cell Signaling Technology, Inc.) and β-actin (1:10000 mouse, Sigma-Aldrich). IRDye 800CW goat anti-rabbit and IRDye 680CW goat anti-mouse were used as secondary antibodies. Blots were imaged using LiCor Odyssey image system.
10
Neuronal Markers Immunohistochemistry
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Following the previously described cell culture conditions, samples were collected at 7 days fixed in a 10% formalin solution and kept at 4 °C until further used for staining procedures. Samples were incubated with beta III Tubulin (anti-beta III Tubulin polyclonal antibody, 1:100 dilution; Abcam, Cambridge, UK), GAP-43 (rabbit GAP43 polyclonal antibody, 1:200 dilution; Thermo Fisher Scientific, Bleiswijk, The Netherlands), NF200 (mouse anti-neurofilament 200 monoclonal antibody, 1:40 dilution; Sigma, Darmstadt, Germany) and Synapsin 1 (rabbit anti-synapsin I polyclonal antibody, 1:200 dilution; Abcam; Cambridge, UK) overnight at 4 °C, in a humidified atmosphere after the quenching of endogenous peroxidase activity (0.3% hydrogen peroxide solution; 30 min). R.T.U. VECTASTAIN® Universal ABC Elite® Kit (Vector Laboratories, Burlingame, CA, USA) was used for secondary antibody detection and incubation revealed by using the Peroxidase Substrate Kit (DAB) (Vector Laboratories, Burlingame, CA, USA), according to the manufacturer instructions. The samples were counterstained with hematoxylin for nuclei visualization, mounted in aqueous mounting medium (Sigma, Darmstadt, Germany), and observed in an optical microscope (Leica DM750 microscope).
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