The internal solution used during whole-cell recordings contained biocytin (0.01%) for post hoc identification of the recorded cells. Enkephalin immunostaining allowed for the determination of the MSN subtype from which the recording was done (McGinty, 2007 (link)). Slices were fixed in 4% paraformaldehyde overnight after recording. Fixed slices were washed three times with 1× PBS (each wash for 5 min) and then blocked in 1× PBS containing 1% Triton X-100, 10% normal donkey serum, and 1% bovine serum albumin for 30 min at RT. The slices were incubated with 1° goat pAb to enkephalin (1:200; ab77273, Abcam) in blocking solution overnight at RT. Following three washes in 1× PBS, slices were incubated with Alexa Fluor 488 Donkey anti-goat IgG (1:1000; Invitrogen) and streptavidin Alexa Fluor 594 conjugate (1:1000; Invitrogen) for 3 h at room temperature. Finally, slices were washed three times with PBS, mounted, and coverslipped with Fluoromount-G (Southern Biotech). Stained slices were imaged under a fluorescent microscope to determine colocalization of enkephalin and biocytin.
Streptavidin alexa fluor 594 conjugate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Streptavidin Alexa Fluor 594 conjugate is a fluorescent labeling reagent. It consists of the protein streptavidin conjugated to the Alexa Fluor 594 dye. This conjugate can be used to detect and visualize biotinylated biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg biotin conjugate, by Thermo Fisher Scientific (3 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Streptavidin biotin blocking kit, by Vector Laboratories (2 mentions)
Dig dna labeling mixture, by Roche (2 mentions)
Anti digoxigenin fluorescein fab fragments, by Roche (2 mentions)
Streptavidin magnetic beads, by Thermo Fisher Scientific (2 mentions)
Nuclei ez prep kit, by Merck Group (2 mentions)
Azide peg3 biotin conjugate, by Merck Group (2 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 645, by Jackson ImmunoResearch (2 mentions)
Ab2251, by Merck Group (2 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Anti mouse envision polymer, by Agilent Technologies (2 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Ab77273, by Abcam (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Prolong diamond antifade with dapi, by Thermo Fisher Scientific (1 mentions)
26 protocols using streptavidin alexa fluor 594 conjugate
1
Identification of Enkephalin-Expressing MSNs
2
Visualizing MAP4K4-BioID2 Proteins in DAOY Cells
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To visualize MAP4K4-BioID2 proteins, stably transduced DAOY cells were plated on glass coverslips and incubated with 50 μM biotin for 16 h. The cells were fixed and permeabilized as described in ref. 68 . The fixed cells were incubated with Streptavidin Alexa Fluor 594 conjugate (Invitrogen) for 1.5 h at RT, followed by incubation with primary antibodies overnight at 4 °C. Secondary antibodies were incubated for 2 h at RT.
For VASP localization analysis, 2000 LA-EGFP DAOY cells were seeded in a 384 well plate (781090, Greiner Bio-One) coated overnight with 0.07 μg/μl collagen I (5005-B, Cell systems) diluted in 70% EtOH. After seeding, the cells were starved for 16 h and treated with 100 ng/ml bFGF for 15 min. Cells were fixed and incubated with primary and secondary antibodies as described above.
Imaging acquisition was performed using an SP8 confocal microscope (Leica). The list of the primary and secondary antibodies used is provided in Supplementary Table1 . Metadata of microscopy image acquisition can be found in Supplementary Data 4 .
For VASP localization analysis, 2000 LA-EGFP DAOY cells were seeded in a 384 well plate (781090, Greiner Bio-One) coated overnight with 0.07 μg/μl collagen I (5005-B, Cell systems) diluted in 70% EtOH. After seeding, the cells were starved for 16 h and treated with 100 ng/ml bFGF for 15 min. Cells were fixed and incubated with primary and secondary antibodies as described above.
Imaging acquisition was performed using an SP8 confocal microscope (Leica). The list of the primary and secondary antibodies used is provided in Supplementary Table
3
Lectin Labeling of Avian and Human Sialic Acid Receptors
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Chicken and duck muscle cells were grown on 24-well Nunclon flat-bottom plates (Nunc), as they did not proliferate well on glass coverslips. Lectin labeling was performed as previously described (40 (link)). Cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Endogenous biotin activity was blocked by the use of a streptavidin/biotin blocking kit (Vector Laboratories). Cells were then incubated overnight at 4°C in the dark with fluorescein isothiocyanate (FITC)-labeled Sambucus nigra agglutinin (SNA) lectin (human α-2,6-linked sialic acid receptor binding) and biotinylated Maackia amurensis agglutinin II (MAA II) lectin (avian α-2,3-linked sialic acid receptor binding) (Vector Laboratories), both at 10 μg/ml. Subsequently, the cells were washed three times with TBS and incubated for 2 h at room temperature with a streptavidin-Alexa Fluor 594 conjugate (Invitrogen). Cells were washed again three times with TBS and mounted with ProLong Gold antifade reagent with 4′,6′ diamidino-2-phenylindole (DAPI) (Invitrogen). Negative controls were processed without the use of lectin.
4
Immunohistochemical Analysis of IL-33 and mMCP-8 in Excised Eye Tissues
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Excised eye specimens were fixed with 4% (wt/vol) paraformaldehyde and then embedded in paraffin. The tissues were sectioned at 4-μm thickness, and deparaffinized sections were subjected to hematoxylin and eosin (H&E) staining or immunohistochemistry. Immunofluorescence for IL-33 was as described previously7 (link). In brief, sections were incubated with an affinity-purified rabbit anti-mouse IL-33 polyclonal antibody, and bound antibodies were visualized with a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and a Streptavidin, Alexa Fluor® 594 conjugate (Invitrogen, Carlsbad, CA). Immunofluorescence for mMCP-8 was as described previously9 (link). Sections were incubated with anti-mMCP-8 antibody (clone TUG8, BioLegend), and bound antibodies were visualized with a Cy3-conjugated donkey anti-rat IgG antibody (WAKO, Osaka, Japan). Following mounting with a ProLong® Diamond Antifade with DAPI (Life Technologies, Gaithersburg, MD), fluorescence images were recorded using a confocal laser scanning microscope LSM780 (Carl Zeiss MicroImaging, Thornwood, NY).
5
Lectin Staining for Influenza Receptors
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Cells were grown on 8-well Lab-Tek II chamber slides (Nunc). Lectin labelling was performed as previously described [15 (link)]. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, after which endogenous biotin activity was blocked using a streptavidin/biotin blocking kit (Vector Laboratories). Cells were incubated with fluorescein isothiocyanate (FITC)-labelled Sambucus nigra agglutinin (SNA), specific for human influenza receptor type SAα2,6-Gal, and biotinylated Maackia amurensis agglutinin II (MAA II) (Vector Laboratories) specific for avian influenza receptor type SAα2,3-Gal overnight at 4 °C, at 10 μg/ml each. After overnight incubation, cells were washed twice with Tris-buffered saline (TBS) and subsequently incubated with streptavidin-Alexa Fluor 594 conjugate (Invitrogen) at room temperature for 2 h. Finally, cells were washed three times with TBS and mounted with ProLong Gold antifade reagent with 4′,6′ diamidone-2-phenylindole (DAPI) (Invitrogen). Neuraminidase derived from Clostridum perfringens (11,585,886,001; Roche) was used at 0.05 U/ml in culture medium for 4 h at 37 °C for the collective removal of SA receptors [16 (link)].
6
Detecting Plasmid Amplification in Cell Lines
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Plasmid pSFVdhfr was used to prepare a probe for detection of the plasmid sequence in clone 12 (DM) and clone 22 (HSR) cells [5 (link)]. Cosmid DNA (c-myc) was used to detect natural amplicons in COLO 320DM and COLO 320HSR cells [6 (link)]. Probes were DIG- or biotin-labeled using the BioPrime DNA Labeling System (Invitrogen) with or without 10×DIG DNA Labeling Mixture (Roche Lifescience Inc.). The probe was hybridized to metaphase chromosome spreads, and hybridized DIG- and biotin-labeled probes were detected using anti-Digoxigenin-Fluorescein Fab fragments (Roche) or Streptavidin Alexa Fluor 594 conjugate (Invitrogen), respectively.
7
Immunofluorescence Imaging of Drosophila Eyes
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Third-instar larval imaginal eye discs and adult eyes were dissected in PBS, fixed in 4% (w/v) paraformaldehyde (PFA) in 0.05% (v/v) TX-100 in PBS for 20 min at RT then permeabilised for 20 min at RT in 0.5% (v/v) TX-100/PBS, before blocking for 30 min in 5% (w/v) BSA in 0.05% TX-100/PBS. Subsequently, fixed and permeabilised discs were incubated overnight with rat anti-HA-biotin high affinity antibody (Clone 3F10, Roche) and mouse anti-CLU antibody (G7), as above. Samples were then incubated overnight in streptavidin Alexa Fluor 594 conjugate (Invitrogen; 1:10,000) and anti-mouse Alexa Fluor 488 conjugate (Invitrogen; 1:1000). All antibodies were diluted in the blocking buffer described above. Tissue was counterstained with TOTO-3 (Invitrogen; 1:10,000) diluted in 0.05% TX-100/PBS to detect nucleic acids.
8
Visualizing AREG and FoxM1 in Parental and Tet-Treated Cells
9
Chromosome Spread and Probe Hybridization Protocol
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Metaphase chromosome spreads were prepared by a standard protocol [21 ]. Chromatin fibers were prepared according to our previously published protocol [5 (link)], which was developed based on the preceding protocol [22 (link)]. DIG-labeled plasmid probes were prepared from pG5 or pKV-AR1 plasmid DNA using the BioPrime DNA Labeling Kit (Invitrogen) and 10× DIG DNA Labeling Mixture (Roche). Biotin-labeled G5 probe was prepared by PCR amplification of the G5 sequence (966 bp) in the pG5 plasmid using the primer set used for the preparation of repeat DNA (see above) and biotin labeling of the product using the Biotin PCR labeling kit (Promokine). The probe was hybridized to metaphase chromosome spreads, and hybridized DIG- and biotin-labeled probes were detected using anti-Digoxigenin-Fluorescein Fab fragments (Roche) or Streptavidin Alexa Fluor 594 conjugate (Invitrogen), respectively. For the flow cytometric analysis to evaluate d2EGFP expression, the cells were resuspended in phosphate buffered saline and analyzed using FACS Calibur (Becton Dickinson Co.).
10
Apicoplast Segregation Assay in Parasites
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Parasites were added to a HFF monolayer and grown for 20 hr before fixation with ice cold methanol for 2 min at room temperature. IFAs were performed using a streptavidin-Alexa Fluor 594 conjugate (Invitrogen) as a marker for the apicoplast. Correct apicoplast segregation was determined in duplicate for three independent assays. At least 228 vacuoles were counted per strain. Counts were performed in a blinded manner. The results were statistically tested with an unpaired t test in GraphPad Prism 7. The data presented are as mean ± s.d.
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