Brain slices were first fixed in 4% PFA in 0.01 M PBS after recordings, then slices were incubated with blocking buffer containing 2% Triton X-100 (Sigma-Aldrich) and either 5% normal goat serum or 5% normal donkey serum in PBS for 1 h at room temperature. To visualize biocytin-filled cells and confirm their identity, slices were incubated with Alexa Fluor 350-conjugated streptavidin (1:200, S11249, ThermoFisher Scientific) and either chicken anti-GFP antibody (1:300, GFP-1020, Aves) for Hcrt-GFP neurons or rabbit anti-DsRed antibody (1:1,000, 632496, Clontech) for Gad2–mCherry neurons in PBS containing 1% Triton X-100 and 5% corresponding serum overnight. Secondary antibodies used were either Alexa Fluor 488-conjugated goat anti-chicken antibody (1:1,000, A11039, ThermoFisher Scientific) or Alexa Fluor 568-conjugated donkey anti-rabbit antibody (1:1,000, A10042, ThermoFisher Scientific). Images were acquired using a Zeiss Cell Observer and Zeiss 510 confocal microscope.
Alexa fluor 350 conjugated streptavidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 350-conjugated streptavidin is a fluorescent-labeled protein that binds to biotin. It is used in various biological applications, such as immunoassays and cell-based assays, to detect and visualize biotinylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (1 mentions)
510 confocal microscope, by Zeiss (1 mentions)
Cell observer, by Zeiss (1 mentions)
Alexa fluor 568 conjugated donkey anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti chicken antibody, by Thermo Fisher Scientific (1 mentions)
Lionheart fx automated microscope, by Agilent Technologies (1 mentions)
Ctan software, by Bruker (1 mentions)
2 protocols using alexa fluor 350 conjugated streptavidin
1
Visualizing Biocytin-Filled Neurons
2
Biotin Stability in PPy-Coated PVDF Fibers
Check if the same lab product or an alternative is used in the 5 most similar protocols
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In order to determine incorporation biotin into, and subsequent stability of biotin within the PPy coating; the PPy-coated PVDF fibers were maintained in phosphate-buffered saline (PBS) at 37 °C for two weeks. Samples were extracted from PBS after 0, 7 and 14 days of incubation and treated with Alexa Fluor™ 350 conjugated streptavidin (Thermofisher, Waltham, MA,USA) for 15 min, rinsed three times with PBS, and imaged using a Lionheart FX Automated microscope (BioTek Instruments Inc., Winooski, VT). Biotin incorporated in the PPy coating binds covalently to Streptavidin and the fluorescent intensity of the conjugated Alexa Fluor™ 350 in PPy coating was then used as an indicator of residual biotin in the PPy coating. The fluorescent intensity from the microscopy images was quantified using CTAn software (v1.17, Bruker BioSpin, Billerica, MA) to analyze biotin stability in the PPy coating over 14 days. This analysis was performed for six samples per group (time point).
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