Single- or dual-color fluorescent in situ hybridizations were carried out as previously detailed (Barske et al., 2018 (link)). Published probes used in this study include barx1 (Barske et al., 2016 (link)), dlx2a (Akimenko et al., 1994 (link)), jag1b (Zuniga et al., 2010 (link)), and sox9a (Yan et al., 2002 (link)). The fgfr2 probe was a gift from S. Paul (UCLA). Partial cDNAs for cdh2, lhx6, lhx8a, ncam1a, and pax9 were amplified and cloned into pCRBlunt-II-TOPO (Life Technologies), then sequence-verified, linearized, and used as templates for in vitro transcription with Sp6 or T7 polymerase (Roche) (Table S1 ). Immunostaining was performed separately or following in situ hybridization, using chick anti-GFP (Abcam ab13970, 1:300), anti-phospho-Histone H3 (Ser10) (Sigma 06–570, 1:500), or anti-Alcama (DSHB Zn8, 1:2000) with Alexa dye-conjugated secondary antibodies (1:300, Thermofisher). Alcian Blue and Alizarin Red staining of larvae and adult facial skeletons was performed as described (Ullmann, 2011 ; Walker and Kimmel, 2007 (link)). For all mutant/transgenic analyses, a minimum of n = 2 or 3 individuals with the genotype in question were imaged and evaluated.
Alexa dye conjugated secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa-dye-conjugated secondary antibodies are fluorescently-labeled antibodies used in immunoassays and fluorescence-based detection techniques. They are designed to bind to primary antibodies, allowing for visualization and quantification of target proteins or other biomolecules.
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Lab products found in correlation
Vectashield mounting medium, by Vector Laboratories (10 mentions)
Tissue tek oct compound, by Sakura Finetek (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (5 mentions)
Crest coated slides, by Matsunami (4 mentions)
Vectashield mounting medium containing dapi, by Vector Laboratories (4 mentions)
Lsm 510, by Zeiss (4 mentions)
Mowiol mounting medium, by Merck Group (3 mentions)
N propyl gallate, by Merck Group (3 mentions)
Cytospin, by Thermo Fisher Scientific (3 mentions)
Fluoroshield, by Merck Group (3 mentions)
Ab13970, by Abcam (2 mentions)
Vibratome, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fitc conjugated lectin, by Merck Group (2 mentions)
Cm1850 uv, by Leica (2 mentions)
Fv1200, by Olympus (2 mentions)
Mas gp typea coated slides, by Matsunami (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hrp conjugated anti mouse igg antibody, by Southern Biotech (2 mentions)
Anti rabbit protein a hrp conjugated antibody, by Southern Biotech (2 mentions)
47 protocols using alexa dye conjugated secondary antibody
1
Multimodal Characterization of Craniofacial Development
2
Immunolabeling and Quantification of Neuronal Markers in Mouse Brain
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Mice were deeply anesthetized and intracardially perfused with 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4), and brains were fixed overnight. Each brain was sliced (50 µm) coronally with a vibratome (Leica), and the sections including the LV wall were immunolabeled as previously described109 . The following primary antibodies were used: DRD1 (Sigma-Aldrich, D2944, 1:200), DRD2 (Millipore, Burlington, MA; AB5084p, 1:200), acetylated tubulin (Sigma-Aldrich, T6793, 1:1000), γ-tubulin (Sigma-Aldrich, T5192, 1:1000), β-catenin (BD Transduction Lab, San Jose, CA; 610153, 1:500), GFP (Abcam, ab13970, 1:1000), cleaved caspase-3 (Cell Signaling, Danvers, MA; 9661S, 1:250), Ki67 (Abcam, ab15580, 1:500), BrdU (Abcam, ab6326, 1:200), doublecortin (Abcam, ab18723, 1:1000). Appropriate Alexa dye–conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA; 1:1000) were used to detect primary antibody binding. DAPI (Invitrogen, Carlsbad, CA) was used as the nuclear counterstain. For cell proliferation in the SVZ or tracking neuronal migration from the SVZ to the olfactory bulb, BrdU (100 mg/kg) was injected three times at 2-h intervals. Brains were collected either the next day for SVZ analysis or 21 days later for neuronal migration analysis. Measurements of basal body displacement were performed in a blind manner, without prior knowledge of the mouse genotype.
3
Immunohistochemical Analysis of VIPR1 in PV+ Neurons
4
Immunohistochemical Analysis of Mammalian Tissues
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IHC in sections of sugar glider explants, transgenic mouse skin, and bat plagiopatagium was performed using anti-KRT14 (1:1000; BioLegend, 905303), anti-WNT5A (1:100; BiossUSA, bs-1948R), anti-Ki67 (1:100; Abcam, ab15580), and anti-SOX9 (1:250; Millipore-Sigma, AB5535). Reactions were visualized with horseradish peroxidase (HRP)–streptavidin and the 3-amino-9-ethylcarbazole (AEC) Substrate Kit (Vector Laboratories, SK4200) or Alexa dye–conjugated secondary antibodies (Thermo Fisher Scientific). Control tissue was incubated with PBS instead of primary antibodies. Cell nuclei were stained with DAPI. Sections were examined using an A1R-STED confocal microscope and an NiE upright microscope (Nikon). All images are representative of at least three individuals per stage. IHC of the mouse pinna was performed using the rabbit monoclonal cytokeratin 14 (K14) antisera (SP53; Abcam, ab119695) for 8 hours at 4°C, followed by incubation with goat anti-rabbit immunoglobulin H&L Alexa Fluor 555 (Abcam, ab150078) for 1 hour at room temperature.
5
Immunofluorescence Staining of HBEC Cells
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Fully differentiated HBEC cultures were fixed in -20°C methanol for 10 min, blocked in 10% normal horse serum and 0.1% Triton X-100 in PBS and incubated with primary antibodies (anti-PON2 ab96553, and anti-COX IV ab62164) overnight at 4°C, then with Alexa dye conjugated secondary antibodies (Thermo Fisher) for 30 min at room temperature. Samples were mounted in Mowiol mounting medium containing 2% N-propyl gallate (Sigma) and imaged on a Zeiss LSM 900 confocal microscope (Zeiss).
6
Wholemount Immunofluorescence and SEM Imaging
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For wholemount immunofluorescence, HNECs were fixed in −20°C methanol or 4% paraformaldehyde for 10 min as previously described (31 (link)). Transwell filters were cut out of the plastic supports and placed in a humid chamber for staining. Samples were blocked in 10% normal horse serum and 0.1% Triton X-100 in PBS and incubated with primary antibodies for 1–2 h, then with Alexa dye conjugated secondary antibodies (Thermo Fisher) for 30 min at room temperature. Filters were mounted in Mowiol mounting medium containing 2% N-propyl gallate (Sigma). Samples were imaged with a Leica SP8 or Zeiss LSM 900 confocal microscope. For antibodies and fixation conditions, see Supplemental Table S1. For SEM, Transwell filters were fixed in 2% glutaraldehyde, 4% paraformaldehyde in 0.1 M NaCacodylate buffer, pH 7.4 at 4°C overnight. Samples were osmicated, dehydrated, dried with a Tousimis Autosamdri-815 critical point dryer. Samples were mounted luminal side up, sputter coated with 100 Å layer of Au/Pd. Images were acquired with a Hitachi S-3400N VP-SEM microscope operated at 10–15 kV, with a working distance of 7–10 mm and using secondary electron detection.
7
Immunolabeling of Vascular and Cell Markers
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The primary antibodies used for immunolabeling included rat anti-CD31/PECAM-1 (BD Biosciences #553370, RRID:AB_394816), rabbit anti-RFP (Rockland #600-401-379, RRID:AB_2209751), goat anti-Perilipin (Abcam #ab61682, RRID:AB_944751), rat anti-CD11b (AbD Serotec #MCA74EL, RRID:AB_2129277), rabbit anti-Tyrosine hydroxylase (Millipore #AB152, RRID:AB_390204), chicken anti-GFP (Aves Labs #GFP-1010, RRID:AB_2307313), and rat anti-CD105/Endoglin (BioLegend #120402, RRID:AB_961070). In addition, Alexa dye-conjugated secondary antibodies were obtained from Thermo Fisher Scientific.
8
Immunofluorescence Labeling of Mouse Trachea
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MTEC cultures and mouse tracheas were fixed at −20°C in ice cold methanol for 10 min. For wholemount labeling, tracheas were opened longitudinally and pinned luminal side up onto Sylgard-184 elastomer (Ellsworth Adhesives) slabs. Samples were blocked in 10% normal horse serum and 0.1% Triton X-100 in PBS and incubated with primary antibodies for 1–2 h, then with Alexa dye conjugated secondary antibodies (Thermo Fisher) for 30 min at room temperature. Samples were mounted in Mowiol mounting medium containing 2% N-propyl gallate (Sigma). Specimens were imaged with a Leica SP8 confocal microscope. For antibodies, see Supplementary Table S1 . We previously described the generation and testing of the Pk1 and Pk2 antibodies (Deans et al., 2007 (link); Bassuk et al., 2008 (link)).
9
Immunofluorescent Staining of PAX6 in Cells
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Cells were washed twice with PBS, and fixed with PFA (4% w/vol, ThermoFisher, 28906) for 10min at room temperature (RT). PFA was removed, and cells were washed three times with PBS, incubated for 15min at RT with PBS + 0.1% Triton-X, and blocked with PBS + 5% goat serum for 1h at RT. Cells were incubated with primary PAX6 (1:100 ThermoFisher, 14-9914-95) antibody in PBS + 5% goat serum overnight at 4°C. On the next day, cells were washed three times with PBS and incubated with Alexa-dye conjugated secondary antibodies (ThermoFisher) diluted 1:500 in PBS + 5% goat serum at RT form 1h. Cells were washed 3 times with PBS and TO-PRO3 (TO-PRO-3 Iodide (642/661), ThermoFisher T3605) was added to the second wash step.
10
Immunofluorescence Staining of Cell Cultures
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A total of 50,000 cells were plated on 12-mm glass coverslips in a 24-well dish. The next day, cells were fixed with 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.2) for 20 min, washed, and then blocked in 5% BSA + 0.1% saponin (Sigma) in PBS for 1 h. This was followed by overnight staining with primary antibodies at 4 °C, washing with PBS + 0.1% saponin, and 1-h staining with Alexa dye conjugated secondary antibodies (Thermo Fisher Scientific) at room temperature. Coverslips were then mounted on glass slides with Prolong Gold mounting media containing DAPI (Thermo Fisher Scientific). A detailed protocol is available at 10.17504/protocols.io.81wgbym7ovpk/v1 .
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