The immunohistochemistry was performed as described earlier with some modifications48 (link),49 (link). Mouse tissues were fixed in 10% NBF and embedded in paraffin blocks by the Molecular Pathology Core at Columbia University. The deparaffinized sections were subjected to antigen retrieval with 10 mM sodium citrate (pH 6.0), incubated with 0.3% H2O2, blocked with 3% bovine serum albumin (BSA), and then incubated with anti-IR antibodies (CT-3, Millipore, Cat. # MABS65, 1:100) in 3% BSA in PBS with 0.1% Triton X-100 (0.1% PBST) at 4 °C overnight. The slides were washed and incubated with secondary antibodies (Alexa Fluor 568 donkey anti-mouse, Invitrogen, Cat. #A10037, 1:200). The washed slides were stained with 4′,6′-diamidino−2-phenylindole (DAPI). Ten images were randomly taken under x60 magnification with a Leica Thunder Imager (Leica Microsystems Inc.). Images of sections were acquired as a series of 0.2 μm stacks. The cell edges were defined with Image J as described earlier48 (link). The whole cell signal intensity (WC) and intracell cellular intensity (IC) were measured. The plasma membrane signal intensity (PM) was calculated by subtracting IC from WC. Identical exposure times and magnifications were used for all comparative analyses.
Alexa fluor 568 donkey anti mouse
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 568 donkey anti-mouse is a fluorescently labeled secondary antibody. It is designed to detect and visualize mouse primary antibodies in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (13 mentions)
Alexa fluor 488 donkey anti goat, by Thermo Fisher Scientific (4 mentions)
A10037, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 568 donkey anti rabbit, by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Confocal microscope, by Nikon (2 mentions)
Ds ri1, by Nikon (2 mentions)
Vectashield hardset mounting medium, by Vector Laboratories (2 mentions)
A21206, by Thermo Fisher Scientific (2 mentions)
Zen 2011, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Leica thunder imager, by Leica Microsystems (1 mentions)
Rpte cells, by Lonza (1 mentions)
35 protocols using alexa fluor 568 donkey anti mouse
1
Immunohistochemistry Protocol for Tissue Analysis
2
Generating and Characterizing BK Polyomavirus in Renal Proximal Tubular Epithelial Cells
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RPTE cells were obtained from Lonza and used at passage 6 and 7 for all experiments. RPTE cells were grown in renal epithelial basal media supplemented with the REGM bulletkit (Lonza).
BK-Dunlop inserted into a pGEM vector (kindly provided by M. Imperiale) was digested using BamHI, then purified and re-ligated. The re-ligated genome was transfected into a T75 flask of RPTE cells in 5% FCS REGM. After three weeks, the cells were split into three T150 flasks and left for a further three weeks before harvesting. The cells were freeze thawed three times to release the virus and assayed using a fluorescent focus assay (protocols based on [57 (link)]).
The primary antibodies used were PAb597 against SV40 VP1 (kindly provided by W. Atwood), P5G6 against BKPyV VP1 (kindly provided by D. Galloway), ab53977 against SV40 VP1 (Abcam), ab53983 against SV40 VP2 and VP3 (Abcam), PAb416 against SV40 T-antigen (Abcam), H4A3 against LAMP-1 (Developmental Studies Hybridoma Bank) and ab6160 against tubulin [YL1/2] (Abcam). Secondary antibodies used were Alexa Fluor 568 donkey anti-mouse and Alexa Fluor 488 or 568 goat anti-rabbit (Invitrogen). LysoTracker red DND-99 was obtained from Life Technologies.
BK-Dunlop inserted into a pGEM vector (kindly provided by M. Imperiale) was digested using BamHI, then purified and re-ligated. The re-ligated genome was transfected into a T75 flask of RPTE cells in 5% FCS REGM. After three weeks, the cells were split into three T150 flasks and left for a further three weeks before harvesting. The cells were freeze thawed three times to release the virus and assayed using a fluorescent focus assay (protocols based on [57 (link)]).
The primary antibodies used were PAb597 against SV40 VP1 (kindly provided by W. Atwood), P5G6 against BKPyV VP1 (kindly provided by D. Galloway), ab53977 against SV40 VP1 (Abcam), ab53983 against SV40 VP2 and VP3 (Abcam), PAb416 against SV40 T-antigen (Abcam), H4A3 against LAMP-1 (Developmental Studies Hybridoma Bank) and ab6160 against tubulin [YL1/2] (Abcam). Secondary antibodies used were Alexa Fluor 568 donkey anti-mouse and Alexa Fluor 488 or 568 goat anti-rabbit (Invitrogen). LysoTracker red DND-99 was obtained from Life Technologies.
3
Immunofluorescence Staining of Cells
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Cells were fixed with 4% paraformaldehyde in PBS for 1 h at room temperature then permeabilized with 1% Triton X-100 in PBS for 20 min. The cells were blocked with 1% BSA in PBS for 1 h and incubated with primary antibodies overnight at 4 °C. Secondary antibody incubation was performed using Alexa Fluor 568 donkey anti-mouse or Alexa Fluor 488 donkey anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Then, 5 mg/mL DAPI was added to stain the nucleus. The slides were mounted and visualized using a Nikon confocal microscope (Nikon, Melville, NY, USA).
4
Immunofluorescent Staining of Neural Stem Cells
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Immunofluorescent staining of mature or differentiated NSs was performed as described (Golmohammadi et al. 2008 (link)). The NSs were fixed with 4% paraformaldehyde (Amresco, Solon, OH) for 30 min at RT. After one PBS wash, they were blocked in blocking solution [PBS with 0.1% triton100 (Amresco), 5% FBS, and 5% goat serum (ZSGB-Bio, Beijing, China)] for 60 min at 37°C. After that, the NSs were firstly stained with primary antibody and then with secondary antibody in blocking solution, the nuclei were stained with 10 μg/mL Hoechst (1:1000; Beyotime, Jiangsu, China). Primary antibodies: mouse-anti-nestin (1:200; 307501, R&D, Minneapolis, MN), rabbit-anti-SOX2 (1:200; L1D6A2, Cell Signal Technology (CST), Boston, MA), mouse-anti-GFAP (1:200; GA5, CST) and rabbit-anti-ßIII-tubulin (1:200; D65A4, CST); secondary antibodies: Alexa Fluor 568 donkey-anti-mouse (1:1000; Invitrogen/molecular probe) and Alexa Fluor 488 donkey-anti-rabbit (1:1000; Invitrogen/molecular probe). The coverslips were then mounted using GVA mounting medium (ZSGB-Bio).
5
Immunofluorescence for PDAC Cell Characterization
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Immunofluorescence in PDAC cells was performed as described previously [6 (link)], but using 13% or 4.5% of paraformaldehyde and utilizing the following primary and secondary antibodies: Myctag (Cell Signaling, #2272S, 1:200; Cell Signaling Technology, Danvers, MA, USA), HA (Cell Signaling, #2367, 1:100; Cell Signaling Technology, Danvers, MA, USA), pSer21EZH2 (Bethyl IHC-00388, 1:100; Bethyl, Montgomery, AL, USA), ALEXA Fluor 568 donkey anti-mouse (Invitrogen #A10037, 1:500; Carlsbad, CA, USA), and ALEXA Fluor 488 donkey anti-rabbit (Invitrogen #A32790, 1:500; Carlsbad, CA, USA). To assess the HA-NFATc1/pSer21EZH2 colocalization coefficient, 1228 DAPI-stained nuclei were counted in eleven IF pictures to determine the number of nuclei positive for both HA-NFATc1 and pSer21EZH2. H&E staining and IHC were performed as previously described [6 (link)]. Primary antibodies were: EZH2 (mouse: Cell Signaling #5246, 1:100; human: Leica NCL-L-EZH2, 1:300; Leica Biosystems Wetzlar, Germany), NFATc1 (abcam ab25916, 1:200; Cambridge, UK), and pSer21EZH2 (Bethyl IHC-00388, 1:50; Bethyl, Montgomery, AL, USA).
6
Dual Immunofluorescence Labeling of TH and GFP
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Sections were blocked in 10% normal donkey serum for 1 hour and subsequently transferred to the primary antisera (TH: Millipore Ab318, mouse anti-TH, 1:4,000) to incubate overnight at 4 °C. Following primary incubation, tissue was incubated in the dark in secondary antisera against mouse IgG (Invitrogen A10037, Alexa Fluor 568 donkey anti-mouse, 1:500) for 1 hour at room temperature. Tissue was then blocked in 10% normal goat serum for 1 hour at room temperature before being incubated in the primary antisera against GFP (Abcam Ab290, rabbit anti-GFP, 1:100,000) overnight at 4 °C in the dark. Following primary incubation, tissue was incubated in secondary antisera against rabbit IgG (Life Technologies, Grand Island, NY; A21206, Alex Fluor 488 goat anti-rabbit IgG, 1:500) for 1 hour at room temperature. Sections were mounted on subbed slides and coverslipped with Vectashield Hardset Mounting Medium (Vector Laboratories). Images were taken on a Nikon 90i fluorescence microscope with a Nikon DS-Ri1 camera. Figures were produced in Photoshop 7.0. Brightness, saturation, and sharpness were adjusted only as necessary to best replicate the immunostaining as viewed directly under the microscope.
7
Multicolor Immunofluorescence Labeling Protocol
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A 1 in 12 series of sections were blocked in 10% normal donkey serum for 1 hour and subsequently transferred to the primary antisera (NeuN: Millipore Ab377, mouse anti-NeuN 1:1,000) to incubate overnight at 4 °C. Following primary incubation, tissue was incubated in the dark in secondary antisera against mouse IgG (Invitrogen A10037, Alexa Fluor 568 donkey anti-mouse, 1:500) for 1 hour at room temperature. Tissue was then blocked in 10% normal goat serum for 1 hour at room temperature before being incubated in the primary antisera against GFAP (Abcam AB7260, rabbit anti-GFAP, 1:10,000) or Iba-1 (Wako 019-19741, rabbit anti-Iba-1, 1:1,000) overnight at 4 °C in the dark. Following primary incubation, tissue was incubated in secondary antisera against rabbit IgG (LI-COR 925–68021, goat anti-rabbit IRDye 680LT, 1:500) for 1 hour at room temperature. Sections were mounted on subbed slides and coverslipped with Vectashield Hardset Mounting Medium (Vector Laboratories). Images were taken on a Nikon 90i fluorescence microscope with a Nikon DS-Ri1 camera. Figures were produced in Photoshop 7.0. Brightness, saturation, and sharpness were adjusted only as necessary to best replicate the immunostaining as viewed directly under the microscope.
8
Immunofluorescence Staining of Cj Cas9
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Cells were washed in PBS and fixed in 4% paraformaldehyde for 15 min. After several washes in TBST, cells were permeabilized with 0.3% Triton-X-100 for 10 min, blocked with PBS containing 2% BSA 20 min, and incubation with primary antibodies overnight at 4°C. Then cells were treated with secondary antibody for 45 min at room temperature and mount with DAPI after three times washes in TBST. The following antibodies were used in this study: anti-HA-tag antibody (MBL, M180–3) for CjCas9, Alexa Fluor 488 goat anti-mouse (Invitrogen, A-11001), and Alexa Fluor 568 donkey anti-mouse (Invitrogen, A-10037).
9
Immunoblotting and Immunofluorescence Antibody Panel
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The following primary antibodies were used for immunoblotting and immunofluorescence: α-Actin (A5316, Sigma Aldrich), α-ATG3 (3415, CST), α-ATG101 (SAB4200175, Sigma-Aldrich), α-ATG13 (M183-3, MBL; SAB4200100, Sigma-Aldrich), α-ATG14 (PD026, MBL), α-ATG14 pS29 (92340, CST), α-Coilin (PA5-29531, Invitrogen), α-FIP200 (A301-574A, Bethyl), α-GAPDH (ab8245, Abcam), α-GFP (3H9, Chromotek), α-LC3B (2775, CST), α-p62 (GP62-C, PROGEN), α-pICln (sc-393525, Santa Cruz), α-PRMT5 (2252, CST), α-SmB (S0698, Sigma-Aldrich), α-SmD1 (ab79977, Abcam), α-SmD2 (SAB2102257, Sigma-Aldrich), α-SmE (NBP2-43792, Novus), α-SmF (SAB2102258, Sigma-Aldrich), α-SmG (HPA064152, Sigma-Aldrich), α-SMN (clone 2B1, 05-1532, Merck Millipore), α-SNRPB (Y12, MA5-13449, Invitrogen), α-Tubulin (clone B512, T5168, Sigma Aldrich), α-ULK1 (8054; CST), α-ULK2 (ab97695, Abcam), α-WD45 (2823, CST). The detection of proteins was carried out with the following fluorescent secondary antibodies: IRDye 680LT goat α-rabbit, IRDye 680LT goat α-mouse, IRDye 800CW donkey α-rabbit, IRDye 800CW donkey α-mouse, IRDye 800CW goat α-rat. For the detection of proteins in vivo via IF the following secondary antibodies were used: Alexa Fluor 568 donkey anti-mouse (A10037, Invitrogen) and Alexa Fluor 647 donkey anti-rabbit (A31573, Invitrogen).
10
Immunocytochemistry of Fixed and Permeabilized Cells
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Cells were fixed with 4% paraformaldehyde in PBS for 1 hour at room temperature and then permeabilized with 1% Triton X‐100 in PBS for 20 minutes. The cells were blocked with 1% BSA in PBS for 1 hour and incubated with primary antibodies overnight at 4°C. Secondary antibody incubation was performed using Alexa Fluor 568 donkey anti‐mouse, or Alexa Fluor 488 donkey anti‐rabbit antibodies (Invitrogen, Carlsbad, CA, USA) for 1 hour at room temperature. In all, 5 mg/mL DAPI was added to stain the nucleus. The slides were mounted and visualized using a Nikon Confocal Microscope (Nikon, Melville, NY, USA).
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