Cells were harvested and these dissociated single cells were incubated with fluorescein-conjugated primary antibodies or negative isotype control in FACS buffer (0.5% BSA and 0.1% sodium azide in PBS) for 30 min on ice. For detection of lineage cocktail, after staining with biotin-conjugated antibody, cells were stained with anti-biotin-APC-conjugated antibody. After washing, cell sorting and analysis were performed using an FACS Aria Cell Sorter (Becton Dickinson, Franklin Lakes, NJ, USA). All used antibodies are listed in Table S1 . For lectin staining, biotin-conjugated Sambucus nigra (SNA) (EY Laboratories, San Mateo, CA, USA), biotin-conjugated Ricinus communis agglutinin I (RCA120) (Vector Laboratories, Peterborough, UK), biotin-conjugated Lycopersicon esculentum (LEL) (Vector Laboratories), biotin-conjugated wheat germ agglutinin (WGA) (J-Oil Mills, Tokyo, Japan), and TexasRed-conjugated streptavidin (Vector Laboratories) were used. Mean fluorescence intensities (MFIs) were calculated by subtracting the intensities of the negative controls.
Texasred conjugated streptavidin
Manufactured by Vector Laboratories
Sourced in Japan
TexasRed-conjugated streptavidin is a fluorescent conjugate used for the detection and visualization of biotinylated biomolecules. Streptavidin, a tetrameric protein, binds strongly to biotin, allowing for the labeling and tracking of target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria cell sorter, by BD (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Axio imager m2 microscope, by Zeiss (1 mentions)
Blocking buffer, by Vector Laboratories (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Fluorescein isothiocyanate (fitc), by Vector Laboratories (1 mentions)
Fv1000, by Nikon (1 mentions)
Ti2 e a1rhd25, by Nikon (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit immunoglobulin, by Thermo Fisher Scientific (1 mentions)
4 protocols using texasred conjugated streptavidin
1
Multiparametric Flow Cytometry Analysis
2
Immunohistochemical Evaluation of rDPSCs
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P1 rDPSCs pretreated with 10% normal goat serum (Vector Laboratories, Burlingame, CA) were incubated with anti-nitrotyrosine rabbit IgG (1:100; Millipore, Billerica, MA) at 4 °C overnight, and then incubated with goat biotinylated anti-rabbit IgG (1:200; Vector Laboratories) for 60 min. After treatment with 10% non-immune goat serum for 30 min, the samples were incubated with anti-iNOS rabbit IgG (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight. Then, they were incubated with fluorescein-labeled goat anti-rabbit IgG (1:50; Thermo Fisher Scientific) and Texas Red-conjugated streptavidin (1:100; Vector Laboratories) for 60 min. The immunohistochemical specificity of these antibodies was evaluated in previous studies14 (link),36 (link),37 (link). Finally, after washing, sections were mounted in VECTASTAIN anti-fade medium with 4ʹ,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). The sections were observed under an Axio Imager M2 microscope (Carl Zeiss Microscopy, Oberkochen, Germany).
3
Immunocytochemistry Protocol for Cellular Analysis
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Cells were fixed in 10% (v/v) formaldehyde/PBS at 4°C for 1 hour. Cells were permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 min at room temperature and blocked with 1% (w/v) bovine serum albumin in PBS with 0.1% (v/v) Tween 20 (PBST) for 1 hour at room temperature. Following blocking, the relevant primary antibodies (Table 2 ) were incubated with cells in blocking buffer overnight at 4°C. Cells were washed three times in PBST and incubated with biotinylated secondary antibodies in blocking buffer (1:50; Vector Laboratories) for 1 hour at room temperature. Cells were again washed three times in PBST and incubated with fluorescein isothiocyanate– or Texas Red–conjugated streptavidin in blocking buffer (1:50; Vector Laboratories). Where appropriate, cell F-actin was labeled through 1-hour incubation at room temperature with rhodamine-conjugated phalloidin (1:1000 in blocking buffer). Nuclei were stained using VECTASHIELD mountant with 4′,6-diamidino-2-phenylindole nuclear stain (Vector Laboratories).
4
Visualizing Sea Star Attachment Footprints
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Footprints were either obtained by turning individual sea stars on their backs and letting them attach strongly to a glass slide placed on their tube feet, or by allowing sea stars to walk over a glass slide. In both cases, the sea stars and glass slides were submerged in seawater. Footprints were fixed in 4% (w/v) PAF in PBS solution, rinsed in PBS solution, dehydrated in ethanol and stored in 70% ethanol. Upon use, they were rehydrated in distilled water followed by Tris-buffered saline (25 mmol l−1 Tris, 125 mmol l−1 NaCl, pH8.0) containing 0.05% (v/v) Tween-20 (TBS-T) and submitted to the immunolabelling method detailed earlier, without the antigen retrieval step. Double immune-labelling was performed with the polyclonal anti-Asterias rubens Sfp1β [6 (link)] and the lectin WGA. This antibody was diluted 1 : 100 and the lectin WGA was diluted at a concentration of 25 µg ml−1 in TBS-T containing 3% (w/v) bovine serum albumin (TBS-T-BSA) [6 (link),13 (link)]. Alexa Fluor 488-conjugated goat-anti-rabbit immunoglobulins (Invitrogen) were diluted 1 : 200 and Texas-Red-conjugated streptavidin (Vector Laboratories) was diluted 1 : 100 in TBS-T-BSA. Footprints were observed by using an Olympus FV1000 or Nikon TI2-E-A1RHD25 a confocal microscope.
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