Biocytin (0.2%) was introduced into cells during whole-cell recordings. After recording, slices were fixed overnight at 4°C in phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA; cat# 15714-S, Electron Microscopy Sciences, Hatfield, PA). Slices were then transferred to PBS and stored for up to 2 weeks at 4°C. After permeabilization with 0.3% Triton X-100 (cat# BP151-500; Fisher Scientific, Pittsburgh, PA) in PBS for 2 h at room temperature, slices were incubated in PBS containing Alexa 633-conjugated streptavidin (final concentration 1 µg/ml; cat #S-21375; Invitrogen, Grand Island, NY) overnight at 16°C. Slices were cryopreserved in PBS containing 30% sucrose, and then resectioned at 100–150 µm thickness with a sliding freezing microtome (HM430, Thermo Scientific, Waltham, MA). After staining with Neurotrace 435/455 (1:100 in PBS; cat #N21479, Invitrogen) and mounting on Colorfrost Plus slides (cat #99-910-11, Fisher Scientific) using Vectashield HardSet Mounting Medium (NC9029228, Fisher Scientific), sections were imaged with a Fluoview FV-1000 confocal imaging system (Olympus, Center Valley, PA) with a 25× objective (XLPL25XWMP, Olympus, Tokyo, Japan).
Alexa 633 conjugated streptavidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa-633-conjugated streptavidin is a fluorescent labeling reagent. It is a complex of the protein streptavidin and the fluorescent dye Alexa Fluor 633. This reagent can be used to detect and localize biotinylated molecules in various applications such as immunoassays, flow cytometry, and microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview fv 1000 confocal imaging system, by Olympus (2 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Neurotrace 435 455, by Thermo Fisher Scientific (1 mentions)
Colorfrost plus slides, by Thermo Fisher Scientific (1 mentions)
Xlpl25xwmp, by Olympus (1 mentions)
Hm430, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa 568, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Tcs sp8 laser scanning microscope, by Leica (1 mentions)
Biocytin, by Merck Group (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Neurolucida 360, by MBF Biosciences (1 mentions)
Axo imager z2, by Zeiss (1 mentions)
Anti fading mounting medium, by Agilent Technologies (1 mentions)
5 protocols using alexa 633 conjugated streptavidin
1
Biocytin Labeling and Confocal Imaging
2
Biocytin-based Morphological Reconstruction
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During electrophysiological experiments, recorded SOM-YFP cells were filled with biocytin for post-hoc morphological reconstruction. After recording, slices were fixed overnight at 4°C in 0.1 M phosphate-buffered saline (PBS) containing 4% paraformaldehyde. After several washes in PBS, and 2 h permeabilization with 0.3% Triton X-100 in PBS at room temperature, slices were incubated overnight at 16°C in PBS with Alexa 633-conjugated streptavidin (final concentration 1 μg/mL, catalogue no. S-21375; Invitrogen). Slices were cryopreserved in 30% sucrose containing PBS and then re-sectioned at 100–150 μm thickness using a sliding freezing microtome (HM430; Thermo Scientific, Waltham, MA, USA). After staining with Neurotrace 435/455 (1:100 in PBS) and mounting on gelatin-coated slides in Vectashield (catalogue no. H-1400; Vector Laboratories), sections were imaged with a Fluoview FV-1000 confocal imaging system (Olympus) with 4x, 25x, and 60x objectives. Tiled confocal stacks (800 × 800 pixels; 0.2 μm z-step) of SOM-YFP cells were flat projected, rotated and cropped in PhotoShop 13.0 or ImageJ for display.
3
Fluorescent Labeling of Biocytin-Filled Neurons
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Tissue cultures were fixed in a solution of 4% (w/v) PFA and 4% (w/v) sucrose in 0.01 m PBS for 1 h. The fixed tissue was incubated for 1 h with 10% (v/v) NGS and 0.5% (v/v) Triton X-100 in 0.01 m PBS. Biocytin (Sigma Millipore, catalog #B4261) filled cells were stained with Alexa-488-, Alexa-568-, or Alexa-633-conjugated streptavidin (Thermo Fisher Scientific; 1:1000; in 0.01 m PBS with 10% NGS and 0.1% Triton X-100) for 4 h, and DAPI (Thermo Fisher Scientific) staining was used to visualize cytoarchitecture (1:5000; in 0.01 m PBS for 15 min). Slices were washed, transferred, and mounted onto glass slides for visualization. Transfected and streptavidin-stained granule cells were visualized with a Leica Microsystems TCS SP8 laser scanning microscope with 20× (NA 0.75), 40× (NA 1.30), and 63× (NA 1.40) oil-submersion objectives. Outer molecular layer segments were imaged with higher scan zoom, and spine densities were determined as described previously (Hick et al., 2015 (link)).
4
Biocytin-based interneuron classification
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Neurons were passively filled with biocytin in the whole-cell configuration. Following recordings, the pipette was carefully retracted, and the acute slice was placed in a petri dish between filter papers. Slices were fixed overnight with 4% PFA in PBS. Biocytin was revealed by treating the slices with Triton (1%) and incubating overnight in an Alexa-633 conjugated streptavidin (1:200, ThermoFisher Scientific). The following day, slices were mounted on microscope slides with ProLong Gold (ThermoFisher Scientific). Images were acquired on a Zeiss confocal system (Axo Imager.Z2). Anatomical tracings were performed in Neurolucida 360 (2.70.1, MBF Bioscience) on a personal computer.
For anatomical classification, the axonal length in the dendritic layers (strata oriens and radiatum) and in the somatic layer (stratum pyramidale) were quantified in Neurolucida. For each cell, axonal length was measured using Neurolucida 360. The axonal length in the somatic or dendritic layers were then normalized to the total axonal length for each cell. Using this dataset, K-means clustering analysis in Python was used to cluster interneurons in two groups.
For anatomical classification, the axonal length in the dendritic layers (strata oriens and radiatum) and in the somatic layer (stratum pyramidale) were quantified in Neurolucida. For each cell, axonal length was measured using Neurolucida 360. The axonal length in the somatic or dendritic layers were then normalized to the total axonal length for each cell. Using this dataset, K-means clustering analysis in Python was used to cluster interneurons in two groups.
5
Biocytin-Filled Cell Visualization
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For mEPSC recordings, biocytin-containing internal solution was used as described above. After recording, tissue cultures were fixed in a solution of 4% (w/v) paraformaldehyde (PFA) and 4% (w/v) sucrose in 0.01 M PBS for 1 h at room temperature. After washing with 0.01 M PBS, the fixed tissue was incubated for 1 h at room temperature in a blocking solution consisting of 10% (v/v) normal goat serum (NGS) and 0.5% (v/v) Triton X-100 in 0.01 M PBS. Biocytin-filled cells were stained with Alexa-633 conjugated Streptavidin (ThermoFisher Scientific, Waltham, MA, USA, Cat# S21375) in a dilution of 1:1000 in 0.01 M PBS with 10% NGS and 0.1% Triton X-100 overnight at 4 °C. DAPI staining was used to visualize cytoarchitecture (1:2000; in 0.01 M PBS for 15 min). Slices were then washed 3 times in 0.01 M PBS, transferred and mounted onto glass slides with anti-fading mounting medium (DAKO) for visualization.
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