The samples were prepared as previously reported,[55 ] with slight modifications. Isolated EVs (PalmReNL‐sEVs: 6 × 107 EVs/µL, PalmReNL‐m/lEVs: 7 × 107 EVs µL−1) were fixed in 1% paraformaldehyde. A formvar‐coated gold grid was kept in a saturated water environment for 24 h and placed on a 50 µL aliquot of EV solution, and allowed to incubate for 20 min while covered. Next, samples were washed and blocked by placing each one face down on top of a 100 µL droplet of the following solutions: PBS (2 ×, 3 min), PBS / 50 mm Glycine (4 ×, 3 min), PBS / 5% BSA (1 ×, 10 min). A 1:100 dilution of anti‐RFP antibody (Rockland Immunochemicals, 600‐401‐379) in 5% BSA/PBS was used for labeling (1 h), followed by six washes in PBS/0.5% BSA. Samples were incubated in a 1:50 dilution of donkey anti‐rabbit immunogold conjugate (Jackson ImmunoResearch, 711‐205‐152) in 5% BSA/PBS (20 min) and washed in PBS (6×) and water (6×). The samples were negative stained with 1% uranyl acetate. Excess uranyl acetate was removed by contacting the grid edge with filter paper and the grid was air‐dried. Samples were observed using a JEOL 1400 Flash Transmission Electron Microscope equipped with an integrated Matataki Flash sCMOS bottom‐mounted camera. The 1400 Flash was operated at 100 kV.
Anti rfp antibody
Manufactured by Rockland Immunochemicals
Sourced in United States
The Anti-RFP antibody is a laboratory reagent used to detect and quantify the presence of the Red Fluorescent Protein (RFP) in various biological samples. This antibody specifically binds to RFP, allowing for its identification and measurement in various experimental and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Texas red, by Jackson ImmunoResearch (2 mentions)
Antibodies to β1 integrin 12g10, by Abcam (2 mentions)
Mt1 mmp antibody, by Merck Group (2 mentions)
Fitc phalloidin, by Jackson ImmunoResearch (2 mentions)
Af647 phalloidin, by Jackson ImmunoResearch (2 mentions)
Prfp c rs vamp3 shrna containing sequence 3764, by OriGene (2 mentions)
Prfp c rs scrambled shrna, by OriGene (2 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
α tubulin antibody, by Merck Group (2 mentions)
β1 integrin antibody aiib2, by Developmental Studies Hybridoma Bank (2 mentions)
Alexa647 conjugated donkey anti rabbit secondary antibody, by Jackson ImmunoResearch (2 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fv1200 confocal, by Olympus (1 mentions)
Clone mec 13, by BD (1 mentions)
Gaasp pmt detectors, by Olympus (1 mentions)
8 protocols using anti rfp antibody
1
Characterization of Extracellular Vesicles by TEM
2
Antibody Characterization for Integrin, VAMP, and MMP Studies
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β1-integrin antibody (AIIB2) was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa (1:200); antibodies to β1-integrin-12G10 (1:200 IF, 1:1000 WB), CD9 (1:1000 WB, 1:50 IP) and HE4 (1:600) were purchased from Abcam; MT1-MMP antibody was from Millipore (1:100 IF, 1:1000 WB); anti-RFP antibody was from Rockland Immunochemicals (1:1200); α-tubulin antibody was obtained from Sigma (1:1000); antibodies to BAP31 (1:1000), VAMP3 (1:750), and CA-125 (1:1000) were purchased from Pierce (Rockford, IL.); MHC-I antibody was a gift from Dr. Janice Blum (IUPUI, Indianapolis, IN.); antibodies to ARF6 were described previously 48 (link). FITC-phalloidin, AF647-phalloidin, TexasRed and Cy5 secondary antibodies were from Jackson ImmunoResearch (1:300); HRP conjugated secondary antibodies were purchased from Cell Signaling (1:5000). GFP-VAMP3 and GFP-VAMP7 were a gift from T. Galli (Institut Jacques Monod, Paris). MT1-MMP-mCherry was a gift from P. Chavrier (Institut Curie, Centre de Recherche, Paris). pRFP-C-RS-VAMP3 shRNA containing sequence 3764 and pRFP-C-RS-scrambled shRNA were purchased from OriGene. Sequence 3764 is AAACGAGCGAGCCAAGTTGAAGAGGAAA.
3
Western Blot Antibody Detection
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In western blot experiments, mouse monoclonal anti‐CD81 (1.3.3.22) antibody (sc‐7637; Santa Cruz, Dallas, TX, USA) and rabbit polyclonal anti‐RFP antibody (600‐401‐379; Rockland, Limerik, PA, USA) were used for detecting CD81 and RFP fusion proteins, respectively, in combination with the secondary antibodies goat‐anti‐mouse IgG‐HRP (sc‐2031; Santa Cruz) and goat‐anti‐rabbit IgG‐HRP (sc‐2030; Santa Cruz).
4
Astrocyte Immunofluorescence Staining
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Primary cultured astrocytes seeded on glass coverslips or brainstem organotypic slices excised from the culture membrane were fixed for 15 min in ice-cold 4% paraformaldehyde (PFA) in phosphate buffer (PBS; pH 7.4). After rinsing in PBS, the samples were incubated for 1 h at room temperature in a solution of 0.3% Triton X-100 (Sigma-Aldrich, T8787) and 10% serum. Samples were incubated overnight at 4 °C with anti-RFP antibody (Rockland Immunochemicals, 600-401-379S; 1:200, Limerick, PA, USA), rinsed in PBS and incubated for 1 h at room temperature with Alexa Fluor 594 antibody (Thermo Fisher Scientific, R37117, Waltham, MA, USA).
5
Immunolabeling and Quantification of Cholinergic Neurons
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At p30, ChAT‐CreXAi14 mice, both control and whisker‐deprived, were anesthetized with isoflurane as described above and administered an intraperitoneal injection of 200 mg/kg sodium pentobarbital until nonreactive. Following trans‐cardiac perfusion with 1xPBS (5 ml/min) and then 10% formaldehyde in 1xPBS (5 ml/min), mouse brains were collected and further incubated overnight at 4°C in 10% formaldehyde and washed three times with 1xPBS for 30 min. The cortices were dissected and used for iDISCO+ clearing as described initially and further updated in https://idisco.info/idisco‐protocol/ . Briefly, during the iDISCO+ protocol, we stained the tdTomato expressing cells with an anti‐RFP antibody (1:1,200 dilution, Rockland, 600‐401‐379) for 5 days followed by Alexa‐647 conjugated donkey anti‐rabbit secondary antibody (1:1,000 dilution, Jackson ImmunoResearch, 711‐605‐152) for 5 days.
Cortex vs striatum quantification: ChAT‐CreXAi14 mice were anesthetized with isoflurane as described above and administered an intraperitoneal injection of 200 mg/kg sodium pentobarbital. Following trans‐cardiac perfusion with 1xPBS (5 ml/min) and then 10% formaldehyde in 1xPBS (5 ml/min), mouse brains were collected and further incubated overnight at 4°C in 10% formaldehyde and washed three times with 1xPBS for 30 min. Brains were cryopreserved in 30% sucrose/ 1xPBS solution overnight and embedded in OCT.
6
Endothelial Cell Staining and Imaging
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Staining was performed essentially as described previously (Yokomizo et al., 2019 (link), 2012 (link)). Staining was performed using anti-PECAM-1/CD31 antibody (Clone MEC 13.3, BD Pharmingen, 1:500) to detect endothelial cells and anti-RFP antibody (Rockland, 1:1000) to detect tdTomato. For immunofluorescent detection, either Cy3 or Alexa-647 conjugated secondary antibodies were used. All confocal microscopy was carried out on an Olympus FV1200 confocal equipped with GaAsP PMT detectors (Olympus).
7
Antibody Characterization for Integrin, VAMP, and MMP Studies
8
Imaging Cleared Brain Tissue with Cre-Expressing Neurons
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At p30 ChAT-CreXAi14 mice, both naïve and whisker deprived, were anaesthetized with isoflurane as above and were administered an intra-peritoneal injection of 200 mg/kg sodium pentobarbital.
Following trans-cardiac perfusion with 1xPBS (5ml/min) and then 10% formaldehyde in 1xPBS (5ml/min), mouse brains were collected and further incubated O.N. at 4°C in 10% formaldehyde and then washed 3 times with 1xPBS for 30 min. The cortices were dissected and used for iDISCO+ clearing as described initially 15 and further updated in https://idisco.info/idisco-protocol/ . Briefly, during the iDISCO+ protocol, we stained the tdTomato expressing cells with an anti-RFP antibody (1:1200 dilution, Rockland, 600-401-379) for 5 days followed by Alexa-647 conjugated Donkey anti-Rabbit secondary antibody (1:1000 dilution, Jackson ImmunoResearch, 711-605-152) for 5 days.
Following trans-cardiac perfusion with 1xPBS (5ml/min) and then 10% formaldehyde in 1xPBS (5ml/min), mouse brains were collected and further incubated O.N. at 4°C in 10% formaldehyde and then washed 3 times with 1xPBS for 30 min. The cortices were dissected and used for iDISCO+ clearing as described initially 15 and further updated in https://idisco.info/idisco-protocol/ . Briefly, during the iDISCO+ protocol, we stained the tdTomato expressing cells with an anti-RFP antibody (1:1200 dilution, Rockland, 600-401-379) for 5 days followed by Alexa-647 conjugated Donkey anti-Rabbit secondary antibody (1:1000 dilution, Jackson ImmunoResearch, 711-605-152) for 5 days.
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