Western Blot:Article Title: Synaptic signatures and disease vulnerabilities of layer 5 pyramidal neurons
Article Snippet: .. rat anti HA (Merck, 11867423001, WB: 1:1000, IHC: 1:1000), Alexa 647-conjugated streptavidin (Thermo Fisher Scientific, S32357, IHC: 1:500), Alexa 647-conjugated PSD95 sdAb FluoTag-X2 (Synaptic Systems N3702-AF647- IHC: 1:500), mouse anti-PSD-95 (NeuroMab clone K28/43, WB: 1:1000), rabbit Homer1 (Synaptic Systems, 160 003, WB: 1:5000, IHC: 1:1000), HRP conjugated streptavidin (Thermo Fisher Scientific, SA10001, WB: 1:5000), mouse anti Gria1 (Millipore, MAB2263, WB: 1:500), chicken anti-GFP (Aveslabs, GFP-1020, IHC: 1:500), rabbit anti Tubulin 3 (Abcam, ab18207, WB: 1:2500), mouse anti Synaptophysin (Sigma, S5768, WB 1:2000), Alexa fluor conjugated secondary antibodies from Invitrogen (1:1000). ..
Article Title: Multiparity exacerbates Aβ accumulation and promotes cellular senescence in a mouse model of amyloidosis.
Article Snippet: Proteins were extracted from cells after 3 days of treatment with 200 nM estradiol (E2) (Sigma Aldrich, E2758), 200 nM progesterone (P4) (MCE, HY-N0437), followed by BCA quantification (Thermo Fisher Scientific, A55864) and western blot analysis. .. Antibodies AR TIC LE IN PR ES S 8 The information of primary antibodies used was as follows: rabbit anti-IBA1 (FUJIFILM Wako Chemicals, 019-19741, PRID: AB_839504), goat anti-IBA1 (Novus, NB100-1028, PRID: AB_3148646, 1:500 for immunofluorescence (IF)), rabbit anti-GFAP (Abcam, ab7260, PRID: AB_305808), rat anti-CD68 (BIO-RAD, MCA1957, PRID: AB_3100585), rat anti-LAMP1 (DSHB, clone 1D4B, PRID: AB_2134500), rabbit anti-PU.1 (CST, 2258S, PRID: AB_2186909), rabbit anti-SYNAPTOPHYSIN (Abcam, ab16659, PRID: AB_443419), rabbit anti-PSD-95 (Invitrogen, 51-6900, PRID: AB_2533914), rabbit anti-BACE1 (CST, 5606S, PRID: AB_1903900), rabbit anti-Amyloid Precursor Protein, C-Terminal (Sigma-Aldrich, A8717, PRID: AB_258409), mouse anti-PSD-95 (1:1000; mouse monoclonal, NeuroMab, 75-028, PRID: AB_2292909), rabbit antiFOSB (1:1000; CST, 2251S, PRID: AB_2106903), rabbit anti-SYNAPTOPHYSIN (1:1000 for WB; Abclonal, A6344, PRID: AB_2766946), rabbit anti-BACE1 (rabbit monoclonal, CST, 5606S, PRID: AB_1903900, 1:1000 for WB, 1:500 for IF), chicken anti-MAP2 (1:5000, Abcam, ab5392, PRID: AB_2138153), rabbit anti-Beta-Actin (1:10000; mouse monoclonal; Proteintech, 66009- 1-Ig, PRID: AB_2687938), rabbit anti-β Tubulin (Proteintech, 10094-1-AP, PRID: AB_2210695, 1:5000 for WB) and mouse anti-GAPDH (ProteinTech, 60004-1-Ig, PRID: AB_2107436, 1:10000 for WB). .. The information of secondary antibodies used was as follows: donkey anti-rabbit IgG (H+L) with Alexa FluorTM 488 (Invitrogen, A-21206, PRID: AB_2535729), donkey anti-rabbit IgG (H+L) with Alexa FluorTM 568 (Invitrogen, A10042, PRID: AB_2534017), donkey anti-rabbit IgG (H+L) with Alexa FluorTM 647 (Invitrogen, A-31573, PRID: AB_2536183), donkey anti-goat IgG (H+L) with Alexa FluorTM 488 (Invitrogen, A-11055, PRID: AB_2534102), donkey anti-goat IgG (H+L) with Alexa FluorTM 568 (Invitrogen, A-11057, PRID: AB_2534104), donkey anti-rat IgG (H+L) with Alexa FluorTM 488 (Invitrogen, A48269, PRID: AB_2893137), donkey anti chicken IgY AR TIC LE IN PR ES S 9 (H+L) highly cross adsorbed secondary antibody, Alexa FluorTM 488 (Invitrogen, A78948, PRID: AB_2921070), all diluted by 10% donkey serum in PBS 1:500 for IF.
Immunohistochemistry:Article Title: Synaptic signatures and disease vulnerabilities of layer 5 pyramidal neurons
Article Snippet: .. rat anti HA (Merck, 11867423001, WB: 1:1000, IHC: 1:1000), Alexa 647-conjugated streptavidin (Thermo Fisher Scientific, S32357, IHC: 1:500), Alexa 647-conjugated PSD95 sdAb FluoTag-X2 (Synaptic Systems N3702-AF647- IHC: 1:500), mouse anti-PSD-95 (NeuroMab clone K28/43, WB: 1:1000), rabbit Homer1 (Synaptic Systems, 160 003, WB: 1:5000, IHC: 1:1000), HRP conjugated streptavidin (Thermo Fisher Scientific, SA10001, WB: 1:5000), mouse anti Gria1 (Millipore, MAB2263, WB: 1:500), chicken anti-GFP (Aveslabs, GFP-1020, IHC: 1:500), rabbit anti Tubulin 3 (Abcam, ab18207, WB: 1:2500), mouse anti Synaptophysin (Sigma, S5768, WB 1:2000), Alexa fluor conjugated secondary antibodies from Invitrogen (1:1000). ..
Gentle:Article Title: Viscoelastic N‑cadherin-like interactions maintain neural progenitor cell stemness within 3D matrices.
Article Snippet: .. Primary antibodieswere incubatedovernight at 4 °C with gentle rocking at the following dilutions: rabbit anti-Ki67 (1:100, Abcam, ab16667), rabbit anti-Sox2 (1:400, Cell Signaling, 23064), mouse anti-Nestin (1:400, BD Biosciences, 556309), mouse anti-N-cadherin (1:400, BD Biosciences, 610920), rabbit anti-nonphospho (active) β-catenin (Ser45) (1:1000, Cell Signaling, 19807), rabbit anti-Axin2 (1:200, Invitrogen, PA5-21093), rabbit anti-cleaved caspase-3 (1:400, Cell Signaling 9661), rabbit anti-S100β (1:500, Proteintech, 15146-1-AP, chicken anti-GFAP (1:500, Aves Labs, GFAP), chicken anti-βIII-tubulin (1:500, Aves Labs, TUJ), rabbit anti-MAP2 (1:500, Sigma Aldrich, ab5622), mouse anti-PSD-95 (1:500, NeuroMab, 75-028), rabbit anti-Synapsin I (1:500, Sigma Aldrich, ab1543). .. Shear rheology was performed using a stress-controlled ARG2 rheometer (TA instruments) with a cone-on-plate geometry (20mm, 1° cone angle).
Article Title: Viscoelastic N‑cadherin-like interactions maintain neural progenitor cell stemness within 3D matrices
Article Snippet: .. Primary antibodies were incubated overnight at 4 °C with gentle rocking at the following dilutions: rabbit anti-Ki67 (1:100, Abcam, ab16667), rabbit anti-Sox2 (1:400, Cell Signaling, 23064), mouse anti-Nestin (1:400, BD Biosciences, 556309), mouse anti-N-cadherin (1:400, BD Biosciences, 610920), rabbit anti-non-phospho (active) β-catenin (Ser45) (1:1000, Cell Signaling, 19807), rabbit anti-Axin2 (1:200, Invitrogen, PA5-21093), rabbit anti-cleaved caspase-3 (1:400, Cell Signaling 9661), rabbit anti-S100β (1:500, Proteintech, 15146-1-AP, chicken anti-GFAP (1:500, Aves Labs, GFAP), chicken anti-βIII-tubulin (1:500, Aves Labs, TUJ), rabbit anti-MAP2 (1:500, Sigma Aldrich, ab5622), mouse anti-PSD-95 (1:500, NeuroMab, 75-028), rabbit anti-Synapsin I (1:500, Sigma Aldrich, ab1543). .. The following day, samples were washed three times with PBS-T for 30 min each, and then secondary antibodies were diluted in antibody dilution solution as follows: goat anti-rabbit Alexa Fluor 488 (1:500, Invitrogen, A-11008), goat anti-mouse Alexa Fluor 594 (1:500, Invitrogen, A-11032), goat anti-chicken Alexa Fluor 647 (1:500, Invitrogen, A-21449).
Immunofluorescence:Article Title: Multiparity exacerbates Aβ accumulation and promotes cellular senescence in a mouse model of amyloidosis.
Article Snippet: Proteins were extracted from cells after 3 days of treatment with 200 nM estradiol (E2) (Sigma Aldrich, E2758), 200 nM progesterone (P4) (MCE, HY-N0437), followed by BCA quantification (Thermo Fisher Scientific, A55864) and western blot analysis. .. Antibodies AR TIC LE IN PR ES S 8 The information of primary antibodies used was as follows: rabbit anti-IBA1 (FUJIFILM Wako Chemicals, 019-19741, PRID: AB_839504), goat anti-IBA1 (Novus, NB100-1028, PRID: AB_3148646, 1:500 for immunofluorescence (IF)), rabbit anti-GFAP (Abcam, ab7260, PRID: AB_305808), rat anti-CD68 (BIO-RAD, MCA1957, PRID: AB_3100585), rat anti-LAMP1 (DSHB, clone 1D4B, PRID: AB_2134500), rabbit anti-PU.1 (CST, 2258S, PRID: AB_2186909), rabbit anti-SYNAPTOPHYSIN (Abcam, ab16659, PRID: AB_443419), rabbit anti-PSD-95 (Invitrogen, 51-6900, PRID: AB_2533914), rabbit anti-BACE1 (CST, 5606S, PRID: AB_1903900), rabbit anti-Amyloid Precursor Protein, C-Terminal (Sigma-Aldrich, A8717, PRID: AB_258409), mouse anti-PSD-95 (1:1000; mouse monoclonal, NeuroMab, 75-028, PRID: AB_2292909), rabbit antiFOSB (1:1000; CST, 2251S, PRID: AB_2106903), rabbit anti-SYNAPTOPHYSIN (1:1000 for WB; Abclonal, A6344, PRID: AB_2766946), rabbit anti-BACE1 (rabbit monoclonal, CST, 5606S, PRID: AB_1903900, 1:1000 for WB, 1:500 for IF), chicken anti-MAP2 (1:5000, Abcam, ab5392, PRID: AB_2138153), rabbit anti-Beta-Actin (1:10000; mouse monoclonal; Proteintech, 66009- 1-Ig, PRID: AB_2687938), rabbit anti-β Tubulin (Proteintech, 10094-1-AP, PRID: AB_2210695, 1:5000 for WB) and mouse anti-GAPDH (ProteinTech, 60004-1-Ig, PRID: AB_2107436, 1:10000 for WB). .. The information of secondary antibodies used was as follows: donkey anti-rabbit IgG (H+L) with Alexa FluorTM 488 (Invitrogen, A-21206, PRID: AB_2535729), donkey anti-rabbit IgG (H+L) with Alexa FluorTM 568 (Invitrogen, A10042, PRID: AB_2534017), donkey anti-rabbit IgG (H+L) with Alexa FluorTM 647 (Invitrogen, A-31573, PRID: AB_2536183), donkey anti-goat IgG (H+L) with Alexa FluorTM 488 (Invitrogen, A-11055, PRID: AB_2534102), donkey anti-goat IgG (H+L) with Alexa FluorTM 568 (Invitrogen, A-11057, PRID: AB_2534104), donkey anti-rat IgG (H+L) with Alexa FluorTM 488 (Invitrogen, A48269, PRID: AB_2893137), donkey anti chicken IgY AR TIC LE IN PR ES S 9 (H+L) highly cross adsorbed secondary antibody, Alexa FluorTM 488 (Invitrogen, A78948, PRID: AB_2921070), all diluted by 10% donkey serum in PBS 1:500 for IF.
Incubation:Article Title: TARP ɣ-8 is a target of ethanol that regulates self-administration and relapse in mice
Article Snippet: .. Next, membranes were incubated overnight at 4°C with one of the following primary antibodies: [rabbit anti-TARP γ-8 (1:8000, S. Tomita, Yale University); mouse anti-CaMKIIα (1:20,000, Millipore #05-532); rabbit antiGluA1(1:1 000, Millipore #04-855); mouse anti-PSD-95 (1:500,000, NeuroMab/Antibodies Inc #75-028); mouse anti-GAPDH (1:10,000; Advanced Immunochemical-1 hr room temperature incubation)]. .. Membranes were washed and then incubated with a secondary antibody (1:10 000; Jackson ImmunoResearch) and all chemiluminescent signals were digitally imaged (ImageQuant LAS 4000, GE Healthcare Life Sciences) after incubation in ECL Select or Prime (GE Healthcare).
Article Title: Viscoelastic N‑cadherin-like interactions maintain neural progenitor cell stemness within 3D matrices
Article Snippet: .. Primary antibodies were incubated overnight at 4 °C with gentle rocking at the following dilutions: rabbit anti-Ki67 (1:100, Abcam, ab16667), rabbit anti-Sox2 (1:400, Cell Signaling, 23064), mouse anti-Nestin (1:400, BD Biosciences, 556309), mouse anti-N-cadherin (1:400, BD Biosciences, 610920), rabbit anti-non-phospho (active) β-catenin (Ser45) (1:1000, Cell Signaling, 19807), rabbit anti-Axin2 (1:200, Invitrogen, PA5-21093), rabbit anti-cleaved caspase-3 (1:400, Cell Signaling 9661), rabbit anti-S100β (1:500, Proteintech, 15146-1-AP, chicken anti-GFAP (1:500, Aves Labs, GFAP), chicken anti-βIII-tubulin (1:500, Aves Labs, TUJ), rabbit anti-MAP2 (1:500, Sigma Aldrich, ab5622), mouse anti-PSD-95 (1:500, NeuroMab, 75-028), rabbit anti-Synapsin I (1:500, Sigma Aldrich, ab1543). .. The following day, samples were washed three times with PBS-T for 30 min each, and then secondary antibodies were diluted in antibody dilution solution as follows: goat anti-rabbit Alexa Fluor 488 (1:500, Invitrogen, A-11008), goat anti-mouse Alexa Fluor 594 (1:500, Invitrogen, A-11032), goat anti-chicken Alexa Fluor 647 (1:500, Invitrogen, A-21449).
Clone Assay:Article Title: Trans-synaptic molecular context of NMDA receptor nanodomains
Article Snippet: .. Primary antibodies: mouse anti-EGFP clone 3E6 (Invitrogen A-11120, RRID: AB_221568), chicken anti-GFP (Invitrogen A-10262, RRID: AB_2534023), rabbit anti-GluN2A (clone JH6097, gift of Rick Huganir), mouse anti-PSD-95 clone K28/43 (Neuromab 75-028, RRID: AB_2877189), rabbit anti-Munc13-1 (Synaptic Systems 126103, RRID: AB_887733), mouse anti-Bassoon clone SAP7F407 (Enzo ADIVAM-PS003, RRID: AB_2313990), GFP-Booster Alexa Fluor 488 nanobody (Chromotek gb2AF488, RRID: AB_2827573), FluoTag-X2 anti-PSD-95 Atto488 single-domain antibody (sdAb) clone 1B2 (Nanotag N3702-At488-L, RRID: AB_3076105) Secondary antibodies: FluoTag-XM-QC anti-mouse IgG kappa light chain single-domain antibody (clone 1A23) conjugated to docking strand F1 or F4 (custom made by Massive Photonics; sdAb from Nanotag), FluoTag-Xm_QC anti-rabbit IgG sdAb (clone 10E10) conjugated to docking site F2 or F3 (custom made by Massive Photonics; sdAb from Nanotag), donkey anti-rabbit Cy3B (conjugated in house as described in methods, IgG Jackson Immunoresearch 711-005-152, RRID: AB_2340585), donkey anti-chicken AlexaFluor488 (Jackson Immuno 703-545-155, RRID: AB_2340375), donkey anti-mouse IgG2A AlexaFluor 647 (Jackson Immuno 115-605-206, RRID: AB_2338917), donkey anti-rabbit AlexaFluor 647 (Jackson Immuno 711-605-152, RRID: AB_2492288), goat anti-mouse Abberior STAR RED (Abberior STRED-1001, RRID: AB_3068620), FluoTag-X4 anti-GFP Abberior Star635p sdAb (mix of clones 1H1 and 1B2) (Nanotag N0304-Ab635p, RRID: AB_3075902), anti-rabbit IgG Abberior STAR580 sdAb (Abberior ST580-1010), FluoTag-X2 anti-mouse Ig kappa light chain AZDye568 (clone 1A23) (Nanotag N1202-AF568, RRID: AB_307956) Validation Antibodies against four proteins were central to the conclusions of the manuscript: GluN2A, GFP, Munc13-1, and PSD-95. ..
Biomarker Discovery:Article Title: Trans-synaptic molecular context of NMDA receptor nanodomains
Article Snippet: .. Primary antibodies: mouse anti-EGFP clone 3E6 (Invitrogen A-11120, RRID: AB_221568), chicken anti-GFP (Invitrogen A-10262, RRID: AB_2534023), rabbit anti-GluN2A (clone JH6097, gift of Rick Huganir), mouse anti-PSD-95 clone K28/43 (Neuromab 75-028, RRID: AB_2877189), rabbit anti-Munc13-1 (Synaptic Systems 126103, RRID: AB_887733), mouse anti-Bassoon clone SAP7F407 (Enzo ADIVAM-PS003, RRID: AB_2313990), GFP-Booster Alexa Fluor 488 nanobody (Chromotek gb2AF488, RRID: AB_2827573), FluoTag-X2 anti-PSD-95 Atto488 single-domain antibody (sdAb) clone 1B2 (Nanotag N3702-At488-L, RRID: AB_3076105) Secondary antibodies: FluoTag-XM-QC anti-mouse IgG kappa light chain single-domain antibody (clone 1A23) conjugated to docking strand F1 or F4 (custom made by Massive Photonics; sdAb from Nanotag), FluoTag-Xm_QC anti-rabbit IgG sdAb (clone 10E10) conjugated to docking site F2 or F3 (custom made by Massive Photonics; sdAb from Nanotag), donkey anti-rabbit Cy3B (conjugated in house as described in methods, IgG Jackson Immunoresearch 711-005-152, RRID: AB_2340585), donkey anti-chicken AlexaFluor488 (Jackson Immuno 703-545-155, RRID: AB_2340375), donkey anti-mouse IgG2A AlexaFluor 647 (Jackson Immuno 115-605-206, RRID: AB_2338917), donkey anti-rabbit AlexaFluor 647 (Jackson Immuno 711-605-152, RRID: AB_2492288), goat anti-mouse Abberior STAR RED (Abberior STRED-1001, RRID: AB_3068620), FluoTag-X4 anti-GFP Abberior Star635p sdAb (mix of clones 1H1 and 1B2) (Nanotag N0304-Ab635p, RRID: AB_3075902), anti-rabbit IgG Abberior STAR580 sdAb (Abberior ST580-1010), FluoTag-X2 anti-mouse Ig kappa light chain AZDye568 (clone 1A23) (Nanotag N1202-AF568, RRID: AB_307956) Validation Antibodies against four proteins were central to the conclusions of the manuscript: GluN2A, GFP, Munc13-1, and PSD-95. ..
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