After 2 days of in vitro culture, chimeric organoids were fixed in 4% PFA for 10 min, permeabilized with 100% cold methanol for 10 min, and incubated with mouse anti-E-cadherin (1:100, BD Biosciences), rabbit anti-WT1 (1:50, Santa Cruz Biotechnology) followed by species-specific secondary antibodies (1:50; Jackson ImmunoResearch Laboratories). To detect human cells, chimeric organoids were incubated with FITC-conjugated anti–Human Nuclear Antigen (HNA, 1:100; Merck Millipore Ltd). Chimeric organoids were mounted with Dako Fluorescence Mounting Medium (DAKO Corporation) and examined using an inverted confocal laser-scanning microscope (LS 510 Meta; Carl Zeiss). 3D images of chimeric organoids were reconstructed using the Axio Vision Imager software (Carl Zeiss).
Species specific secondary antibodies
Manufactured by Jackson ImmunoResearch
Sourced in United States
Species-specific secondary antibodies are laboratory reagents used to detect and quantify target proteins in various biological samples. These antibodies are designed to bind to the primary antibodies that are specific to the species of interest, enabling the visualization and measurement of the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
Rabbit anti wt1, by Santa Cruz Biotechnology (1 mentions)
Ls 510 meta, by Zeiss (1 mentions)
Mouse anti e cadherin, by BD (1 mentions)
Axio vision imager, by Zeiss (1 mentions)
Ab13970, by Abcam (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Cm3050, by Leica (1 mentions)
Anti cd31 af3628, by R&D Systems (1 mentions)
Lsm 880 airyscan confocal microscope, by Zeiss (1 mentions)
Ab72996, by Merck Group (1 mentions)
Ab66138, by Abcam (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (1 mentions)
Complete mini protease inhibitor, by Roche (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 microscope, by Leica (1 mentions)
7 protocols using species specific secondary antibodies
1
Immunostaining of Chimeric Organoids
2
Immunohistochemical Staining Protocol
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The following antibodies were used: chicken anti-GFP (ab13970, Abcam, RRID:AB_300798 , 1:500), goat anti-HRP (Sigma, 1:250), mouse anti-Coracle (c556.9 and c615.16, RRID:AB_1161644 ; developed by R. Fehon, 1:40), mouse anti-E-Cadherin (5D3, RRID:AB_528116 ; developed by B. Gumbiner, 1:100), rabbit anti-dsRed (632496, Clontech, RRID:AB_10013483 :, 1:250), rat anti-Basigin (a kind gift from Anne Ephrussi (Besse et al., 2007 (link)), 1:100), rabbit anti-FLAG (Sigma, 1:100) and mouse anti-βPS integrin (CF.6G11, RRID:AB_528310 ; developed by D. Bower, 1:10). CF.6G11, 5D3, c556.9 and c615.16 were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology. Species-specific secondary antibodies (Jackson Immunoresearch) raised in donkey were used at 1:250. Permeabilization was done with 0.3% Triton-X100 except for rat anti-Basigin staining which required Tween-20.
3
Immunohistochemical Visualization of Bone Vasculature
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Embedded bones were sectioned at 100 µm thickness using a cryostat from Leica (CM3050 S). Sections were hydrated using PBS and permeabilized using 0.5% Triton-X100 in PBS for 15 min at room temperature (RT). Samples were then incubated in blocking solution (0.3% Triton, 1% BSA, 2% donkey serum in PBS) for 30 min at RT prior to primary antibody staining. Primary antibodies were diluted in blocking solution, incubated O/N at 4 °C. Species-specific secondary antibodies (Jackson) diluted in blocking buffer were added and incubated for 2–3 h at RT. Nuclei were stained with Hoechst (1/1000 in PBS) and mounted using FluorSave mounting media. The following primary antibodies were used: rat monoclonal anti-Endomucin (sc-65495, Santa Cruz, diluted 1/200), goat polyclonal anti-CD31 (AF3628, R&D, diluted 1/100), rat monoclonal CD41 FITC‐conjugated (MWReg30, Biolegend, diluted 1/50). All secondary antibodies were from Jackson ImmunoResearch and diluted 1/200.
Images were acquired using a LSM 880 Airyscan Confocal microscope (Zeiss), and processed and analyzed using Zen (Zeiss) and Fiji softwares.
Images were acquired using a LSM 880 Airyscan Confocal microscope (Zeiss), and processed and analyzed using Zen (Zeiss) and Fiji softwares.
4
Immunocytochemistry of Neuromuscular Junctions
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Cultures were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Primary antibodies against NFH (Abcam, ab72996; 1∶1000), synapsin-I (Millipore, AB1543P; 1∶100), activated caspase-3 (BioVision, 3015–100; 1∶100) and phosphorylated (S473) Akt (Abcam ab66138; 1∶200) were diluted in blocking solution (5% donkey serum, 1 mg/ml BSA IgG and protease free, in PBS) and incubated overnight at 4°C. Samples were incubated with species-specific secondary antibodies (Jackson Immunoresearch) for 2 h at room temperature. TMR-conjugated α-bungarotoxin (Sigma, T0195) staining for evidence of acetylcholine receptor (AChR) clusters was performed at 1 µg/ml in PBS for 15 min prior to cell permeabilization and primary antibody incubation. For visualization of nuclei in myotubes, Hoechst 33258 (1 µg/ml) or DAPI was used for live or fixed samples, respectively. After the staining protocol was completed, MFC was peeled from the dish by gently pulling it from the proximal to distal side, to minimize severing of axons. Prolong mounting medium was added and covered with a #1.5, 18×18 mm coverslide.
5
Protein Extraction and Western Blotting from Tissue Samples
6
Whole-Mount Retinal Staining Protocol
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Whole-mounted retinas were stained in accordance with a previous report [21 (link)]. Briefly, enucleated eyes of P7 WT and Exoc3l KO mice
were fixed for 20 min in 4% PFA at RT. A small hole was made in the cornea using a 27 G needle, and a circular incision was introduced using fine scissors. Subsequently, retinal cups were
dissected from the eyes and post-fixed for 30 min in 4% PFA at 4°C. Primary antibodies were rabbit anti-neuron-glial antigen-2 (NG2) (1:200; Millipore-Sigma, St. Louis, MO, USA) and hamster
anti-PECAM1 (1:1000; Abcam, Cambridge, UK ). Secondary antibodies were species-specific secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or streptavidin
coupled to Alexa Fluor dyes (Invitrogen). Nuclei were counterstained with 10 µg/ml Hoechst 33342 (Molecular Probes Inc., Eugene, OR, USA) for 10 min at RT. Whole retinas
were mounted using ProLongTM Gold Antifade Mountant (Thermo Fisher Scientific). Confocal images were acquired under a TCS-SP8 microscope (Leica, Wetzlar, Germany). Fluorescence
was produced using a 552 nm laser for Cy3, a 488 nm laser for Alexa Fluor 488, and a 405 nm UV laser for Hoechst 33342 and 4’,6-diamidino-2-phenylindole (DAPI). Z-stack images were obtained
every 5 µm. The hybrid detector HyD (Leica) was used for signal amplification. Projection images were created by 3D viewer software (Leica) from z-stack images.
were fixed for 20 min in 4% PFA at RT. A small hole was made in the cornea using a 27 G needle, and a circular incision was introduced using fine scissors. Subsequently, retinal cups were
dissected from the eyes and post-fixed for 30 min in 4% PFA at 4°C. Primary antibodies were rabbit anti-neuron-glial antigen-2 (NG2) (1:200; Millipore-Sigma, St. Louis, MO, USA) and hamster
anti-PECAM1 (1:1000; Abcam, Cambridge, UK ). Secondary antibodies were species-specific secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or streptavidin
coupled to Alexa Fluor dyes (Invitrogen). Nuclei were counterstained with 10 µg/ml Hoechst 33342 (Molecular Probes Inc., Eugene, OR, USA) for 10 min at RT. Whole retinas
were mounted using ProLongTM Gold Antifade Mountant (Thermo Fisher Scientific). Confocal images were acquired under a TCS-SP8 microscope (Leica, Wetzlar, Germany). Fluorescence
was produced using a 552 nm laser for Cy3, a 488 nm laser for Alexa Fluor 488, and a 405 nm UV laser for Hoechst 33342 and 4’,6-diamidino-2-phenylindole (DAPI). Z-stack images were obtained
every 5 µm. The hybrid detector HyD (Leica) was used for signal amplification. Projection images were created by 3D viewer software (Leica) from z-stack images.
7
Immunohistochemical Profiling of Placental Cells
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Briefly, tissue was fixed in 4% paraformaldehyde and dehydrated by passing through sequential increases in sucrose before embedding in OCT (Thermo Fisher Scientific). Sections were permeabilized with methanol and rinsed with PBS before incubation in blocking buffer (5% BSA and 0.05% Tween 20). CTBs were labeled with rat anti-cytokeratin 7 (1:100, Damsky et al., 1992 (link)) with: PD-L1 (1:100, Cell Signaling, #13684), p62/SQSTM1 (1:100, Novus Biologicals, #H00008878-M01) or NID1 (1:500, R&D Systems, AF2570) for 1 h at 37°C. After three additional PBS washes, primary antibodies were detected using species-specific secondary antibodies (1:1000, Jackson ImmunoResearch Labs, see Table S4 ). Sections were mounted using Vectashield with DAPI (Vector Laboratories). Images were acquired using a Leica DM5000 B inverted microscope. Antibodies and their sources are described in Table S4 . Tissue sections from at least four placentas were evaluated for each condition.
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