Sections were immunolabeled as previously described (Ellisor et al., 2009 (link)). Sagittal sections from Wnt1-Venus embryos at E8.5, E10.5, and E12.5 were analyzed using an anti-GFP antibody (1:600, Molecular Probes, Cat # A-6455). Adult coronal sections for fate mapping experiments were immunolabeled with an anti-β-galactosidase (β-gal) antibody (1:500, Biogenesis, Cat # 4600-1409 or 1:500, Abcam, Catalog # ab9361-250) to identify Wnt1-derived cells (Ellisor et al., 2009 (link)). In addition, anti-Calretinin (1:5000, Chemicon; Billerica, MA; Catalog # AB1550), anti-Calbindin (1:1000, Swant, Catalog # CB3a), and anti-Parvalbumin (1:1000, Sigma, Catalog # P3088-.2ML) antibodies were used as biomarkers. Secondary antibodies were prepared at 1:500 and include: Alexa 488 (Invitrogen; Cat # A-21206, donkey anti-rabbit IgG; Cat # A-21202, donkey anti-mouse IgG; Cat #A-11055 donkey anti-goat IgG), Dylight 549 (Jackson ImmunoResearch Laboratories; Cat #703-505-155, donkey anti-chicken).
Anti calbindin
Manufactured by Swant
Sourced in Switzerland
Anti-calbindin is a laboratory reagent used for the detection and quantification of the calcium-binding protein calbindin in biological samples. It is a specific antibody that binds to calbindin, allowing researchers to study the distribution and expression of this protein in various tissues and cell types.
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Lab products found in correlation
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Anti neun, by Merck Group (2 mentions)
Multiclamp 700a amplifier, by Molecular Devices (2 mentions)
Alexa fluor 555 anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Anti gfp antibody, by Thermo Fisher Scientific (1 mentions)
Anti calretinin, by Merck Group (1 mentions)
Anti parvalbumin, by Merck Group (1 mentions)
Dylight 549, by Jackson ImmunoResearch (1 mentions)
Laser scanning confocal microscope, by Nikon (1 mentions)
Anti glua1 antibody, by Abcam (1 mentions)
Inverted lsm510, by Nikon (1 mentions)
Donkey anti mouse alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Axio imager m2 fluorescent microscope, by Zeiss (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Chemiblocker, by Merck Group (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
9 protocols using anti calbindin
1
Immunolabeling of Wnt1-Derived Cells
2
Whole-cell and LFP Recordings in Hippocampus
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For LFP recordings, glass microelectrodes (tip diameter ~5–10 μm; resistance: 0.2–0.3 MΩ) were filled with ACSF before use. Whole-cell recordings were performed with borosilicate glass electrodes (2–5 MΩ) filled with (in mM) 120 K-gluconate, 10 HEPES, 10 KCl, 3 Mg-ATP, 5 EGTA, 2 MgSO4, 0.3 Na-GTP and 14 phosphocreatine. The pH was adjusted to 7.4 with KOH. LFP signals in the CA3 pyramidal cell layer were amplified 1,000-fold, filtered (1–8 kHz), and sampled at 20 kHz. Whole-cell and extracellular recordings were performed using a Multiclamp 700A amplifier (Axon Instruments). For parallel double patch-clamp and field recordings, a custom-made two channel extracellular amplifier was used. Cells were routinely loaded with 0.2% biocytin. After recordings, slices were transferred to 4% paraformaldehyde. Biocytin-filled cells were subsequently visualized with streptavidin conjugated with Dy-Light 488. After acquisition of confocal images, neuronal reconstruction was performed with the imageJ package (Schneider et al., 2012 (link)). To better estimate the slice position along the dorso-ventral axis slices were either Nissl stained or stained with anti-NeuN (Millipore) or anti-calbindin (Swant) antibodies followed by the secondary polyclonal antibody anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 555 (TermoFisher), respectively.
3
Quantifying Purkinje Cell Density in Cerebellum
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Brains were removed and immersion-fixed with 4% w/v paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 overnight at 4 °C and either embedded in paraffin or cryoprotected by immersion in 0.1 M sodium phosphate buffer (pH 7.4) containing 30% w/v sucrose. Paraffin sections (10 μm-thick), mounted on poly-L-lysine-coated slides, were immunostained with anti-GluA1 antibody (Abcam). All other tissues were quick-frozen on dry-ice, then 30 μm-thick coronal free-floating cerebellar sections immunostained with either anti-calbindin (Swant) or anti-GLAST antibody as described previously (62 (link)). All quantification, carried out blind to genotype, involved counting the number of Purkinje cells in the anterior (lobules I-V, simplex and crus I) and posterior cerebellum (lobules VI -IX, crus II and flocculonodular lobe, lobule X) from three sections/animal and the counts averaged. Data were pooled from GLAST-/- and β-III+/-/GLAST-/- animals as no phenotype observed in β-III+/- animals (62 (link)). Images were captured with either a Zeiss inverted LSM510 or Nikon confocal laser scanning microscope and co-localization analysis carried out using Image J.
4
Immunohistochemical Analysis of TRPV5 and Calbindin
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Animals were injected with anesthesia cocktail (ketamine/xylazine/acepromazine, 50/5/0.5 mg/kg), and under deep anesthesia animals were perfusion fixed with 4% paraformaldehyde. After cryoprotection in 800 mosmol/L sucrose and freezing in Optimal Temperature Cutting (OCT) compound, 5‐μm sections were cut. Sections were incubated overnight at 4°C with anti‐anti‐TRPV5 (1:100, Alomone) or anti‐calbindin (1:500, Swant). Sections were incubated in secondary antibody at 1:2000 for 1 h at room temperature [Alexa Fluor 488 donkey anti‐rabbit (Life Technologies A21206) or Alexa Fluor 555 donkey anti‐mouse (Life Technologies A31570)]. All sections were mounted with ProLong Diamond Antifade Mountant (ThermoFisher Scientific P36970). Images were captured with a ZEISS AXIO Imager M2 fluorescent microscope.
5
Retinal Cell Immunohistochemistry Protocol
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Retinal sections were stained in 5% ChemiBlocker (MerckMillipore #2170), 0.3% Triton-X-100 in PBS (pH, 7.4) overnight at 4°C using anti-Pde6g/h (Santa Cruz #sc-166350), anticone arrestin (Sigma # AB15282), anti-Pde6b (Invitrogen #PA1722), antisecretagonin (gift from Prof. Dr Ludwig Wagner, University of Vienna, Austria), anticalbindin (Swant #300), antiprotein kinase c alpha (Santa Cruz #sc-8393), and antiglutamic-acid-rich protein (Sigma #MABN2429) primary antibodies. Flatmounts were stained in 5% ChemiBlocker, 3% dimethyl sulfoxide, and 0.3% Triton-X-100 in PBS (pH, 7.4) overnight at 4°C using anti-ionized calcium-binding adapter molecule 1 (Fujifilm Wako #019-19741) and anti-β-catenin (Cell Signaling #8480) primary antibodies or isolectin GS-B4 conjugated fluorescein (Sigma #L2895). Corresponding anti-Rabbit AF488 (Invitrogen #A-11070), anti-Rabbit AF647 (Invitrogen #A-21245), and anti-Mouse AF555 (Invitrogen #A-21425) secondary antibodies were incubated in 3% ChemiBlocker in PBS (pH, 7.4) for 1.5 hours at room temperature. Hoechst 33342 (Invitrogen #H1399) was used to stain nuclei. Samples were fixed with Aqua-Poly/Mount (Polysciences #18606) on Thermo Scientific SuperFrost Plus slides and stored at 4°C.
6
Antibody Detection of Seizure Proteins
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N-terminal antibody against seizure protein 6 (Sez6) was described previously [10 (link), 21 (link)]. For detection of seizure 6-like protein (Sez6L) we used monoclonal antibody 21D9 [19 (link)]. Additionally, immunoblotting of mouse brain tissue lysates was performed using the following antibodies: anti-BACE1 (D10E5; Cell Signaling); anti-APP (22C11; Merck Millipore) for flAPP and sAPPt; anti-sAPPβ (SIG39138; Covance) and anti-actin (Sigma Aldrich). For immunohistochemistry of mouse brain tissue, the following antibodies were used: anti-BACE1 (Epitomics; Abcam), anti-APP (Y188; Epitomics, Abcam), anti-GFAP (Dako), anti-CD45 (BD Biosciences) and anti-calbindin (Swant). Immunocytochemistry of primary mouse cortical neurons was performed using these antibodies: anti-BACE1 (Epitomics; Abcam), anti-EEA1 (Cell Signaling), anti-transferrin receptor (TfR; ThermoFisher Scientific) and anti-LAMP1 (1D4B, Santa Cruz Biotechnology).
7
Whole-cell and LFP Recordings in Hippocampus
8
Immunohistochemical Staining of Brain Tissues
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Thirty µm thick slices of frozen brain, cerebellar, and spinal cord tissue were made on a sliding microtome (HM 430 + fast freezing unit KS 34, Thermo scientific, Waltham, Massachusetts) and subsequently stored at −20°C in antifreeze solution. Staining was performed according to a standard protocol. Sections were incubated with primary antibodies over night at 4°C and with secondary antibodies for 2 hr at RT. The following antibodies and dilutions were used for all stains presented: anti-calbindin (Swant, Marly, Switzerland, 1:5000), anti-GAD65/67 (Merck, Darmstadt, Germany; 1:8000), anti-Neuropeptide Y (Thermo Fisher Scientific; 1:1000), anti-Calretinin (Swant; 1:5000), anti-Somatostatin (Acris Antibodies, Herfordt, Germany; 1:50), anti-Parvalbumin (Swant; 1:5000).
9
Immunofluorescence Staining of Neuronal Markers
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Sections were incubated in blocking solution (5% BSA, 1% Donkey Serum, and 0.5% Triton in DPBS) for at least 1 h at room temperature. Sections were then incubated overnight at 4°C with primary antibodies diluted in blocking solution: anti-Tuj1 (1/500, Biolegend), anti-RSK2 (1/100, Cell Signaling Technology), anti-RPS6 (1/100, Cell Signaling Technology), anti-p-RPS6Ser235-236 (1/100, Cell Signaling Technology), anti-p-RPS6Ser240-244 (1/500, Cell Signaling Technology), anti-RFP (1/500, Abcam), anti-SCG10 (1/1,000, Novus), anti-CTB (1/500, Abcam), anti-vGlut1 (1/1,000, Synaptic System), anti-vGat (1/500, Synaptic System), anti-ChAT (1/100, Merck), anti-Islet1-2 (5 μg/ml, DSHB), anti-Advillin (1/500, Proteintech), anti-TrkA (1/500, Bio-techne), anti-Parvalbumine (1/200, Swant), anti-TrkB (1/500, Bio-techne), anti-Calbindin (1/100, Swant), anti-Somatostatin (1/500, Invitrogen), and anti-PGP 9.5 (1/500, Proteintech). Then, tissues were incubated with the appropriated secondary antibodies (Alexa-Fluor conjugated—Jackson laboratories) diluted in blocking solution at 1/500 for 2 h at room temperature. Slides were mounted with Fluoromount-G Mounting Medium, with DAPI Medium (Invitrogen).
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