Oocytes were fixed on coated coverslips with paraformaldehyde or glutaraldehyde, as described previously (Verlhac et al. 1994 (link), Terret et al. 2003 (link)). After incubation with the anti-SKAP (1:200) or rat monoclonal anti-tyrosinated α-tubulin antibody (YL1/2, 1:100, Abcam) and washes, oocytes were incubated with anti-guinea pig-Cy3 or anti-rat-Cy3 secondary antibodies respectively (Jackson ImmunoResearch Laboratories, Inc.). Coverslips were mounted in Prolong Gold with DAPI (Life Technologies). Image acquisition of fixed oocytes was performed using the SP5/AOBS confocal microscope equipped with a Plan APO 63×/1.4 N.A. objective.
Yl1 2
Manufactured by Abcam
Sourced in Germany
YL1/2 is a mouse monoclonal antibody that recognizes an epitope on the human and mouse Lamin A/C proteins. Lamin A/C are type V intermediate filament proteins that are essential structural components of the nuclear lamina. YL1/2 antibody can be used for the detection of Lamin A/C in various applications such as Western blotting and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions)
Anti guinea pig cy3, by Jackson ImmunoResearch (1 mentions)
Anti p chk1 ser317, by Cell Signaling Technology (1 mentions)
Anti smad3, by Abcam (1 mentions)
Anti chk1, by Santa Cruz Biotechnology (1 mentions)
Anti p300 c 20, by Santa Cruz Biotechnology (1 mentions)
Anti maml1, by Cell Signaling Technology (1 mentions)
Anti patr ser428, by Cell Signaling Technology (1 mentions)
Anti atr, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Anti actin, by Thermo Fisher Scientific (1 mentions)
Anti tubulin, by Cell Signaling Technology (1 mentions)
Anti lamin b1, by Abcam (1 mentions)
Ab4448, by Abcam (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Alexa fluor 555 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Hpa041019, by Atlas Antibodies (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Revert 700 total protein stain, by LI COR (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
8 protocols using yl1 2
1
Immunofluorescence Imaging of Oocytes
2
Antibody Analysis for DNA Damage
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The antibodies used in this study were the following: anti-thymine dimer (Insight Biotechnology), (anti-γH2AX, MABE205, Millipore, Schwalbach, Germany), anti-pATM (ser1981, Rockland), anti-ATR (Santa Cruz), anti-pATR (ser428, Cell Signalling), anti-Chk1 (Santa Cruz), anti-pChk1 (ser317, Cell Signalling), and anti-tubulin (YL1/2, Abcam), FLAG-M5 monoclonal antibody (A2220, Sigma), anti-p300 (C-20, Santa Cruz), anti-tubulin (YL1/2, Abcam), anti-MAML1 (Cell Signalling), anti-SMAD3 (Abcam).
3
Immunostaining of Cellular Structures
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Primary antibodies were used for immuno-staining with the following dilution: anti-Tubulin 1:1000 (Cell Signalling Technology YL1/2), anti-LaminB1 1:1000 (Abcam ab16048) and anti-Actin 1:1000 (Thermo-Fisher PA1-183). Secondary antibodies were used with the following dilution: A488 goat anti-Rabbit 1:1000 (Thermo-Fisher A-11034), A488 donkey anti-Rat 1:1000 (Thermo-Fisher A-21208), and A594 goat anti-Rabbit 1:1000 (Thermo-Fisher A-11037). Dyes were used for cell staining with the following dilution: Phalloidin DyLight 650 1:1000 (Invitrogen 13454309), Hoechst 33342 1:2000 (Sigma-Aldrich 14533), SiR-DNA 100 nm (tebu-bio SC007).
4
Immunofluorescence Staining of Stx16 and Centrosomal Markers
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For Stx16 detection, miRNA-transfected cells were grown on coverslips and then fixed with 2% paraformaldehyde in 0.5% Triton-X-100/PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgSO4). For microtubule staining, the cells were pre-extracted in PHEM/0.5% Triton-X-100 for 5 min before fixation using the fixative above supplemented with 0.2% glutaraldehyde. After rinse with MBST (10 mM MOPS, 150 mM NaCl and 0.05% Tween 20), coverslips were blocked in 20% boiled normal goat serum (bngs)/MBST for 1 h at RT. Primary antibodies used were rabbit anti-STX16 (1 : 250; HPA041019, Atlas antibodies), mouse anti-CETN3 (1 : 500; H00001070-M01, Abnova), rat anti-α-tubulin (1 : 500; YL1/2, ab6160, Abcam) and rabbit anti-pericentrin (1 : 500; ab4448, Abcam), all diluted in 5% bngs/MBST and incubated 1 h at RT. Secondary Alexa Fluor goat anti-mouse 555, goat anti-rabbit 488, 555 and 647, and chicken anti-rat 488 antibodies (Invitrogen, Thermo Fisher Scientific) were used at 1 : 500 concentration and incubated for 1 h at RT. DNA was stained with DAPI (1 : 10 000 in MQ H2O) and coverslips mounted on slides using Vectashield (Vector laboratories, Burlingame, CA, USA).
5
Western Blotting Protein Analysis Protocol
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For western blotting analyses, protein samples were separated in TGX minigels (Bio-Rad, Hercules, CA, USA) and transferred on nitrocellulose membranes with the Trans-Blot Turbo system (Bio-Rad). Total protein was stained with the Revert 700 Total Protein Stain (Li-Cor Biosciences, Lincoln, NE, USA) and scanned with the Odyssey scanner (Li-Cor). For muscle lysates, gels were stained post-blotting with Coomassie blue. Blots were stained with the following primary antibodies: calnexin Rb mAb C5C9 (Cell Signaling Technology, 2679, RRID:AB_2228381); DNAJB2 Rb pAb (Proteintech, 10 838-1-AP, RRID:AB_2277491); DNAJB6 Rb mAb [EPR17122] (Abcam, ab198995, RRID:AB_2924896); histone 3 Rb pAb (Abcam, ab1791, RRID:AB_302613); tubulin Rt mAb YL1/2 (Abcam, ab6160, RRID:AB_305328); V5 Ms mAb (Thermo Fisher, R960-25, RRID:AB_2556564); V5 Rb mAb D3H8Q (Cell Signaling Technology, 13202, RRID:AB_2687461). Detection was performed with fluorescent secondary antibodies using an Odyssey scanner or with HRP-conjugated secondary antibodies visualized by enhanced chemiluminescence. Image analysis was done in Fiji (39 (link)).
6
Immunoblotting with Antibody Characterization
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Rabbit anti-SLBP was a gift from Z. Dominski. Rabbit anti-NELF-A was made by immunizing with a GST fusion of human NELF-A (1–204) and affinity purification. Rat anti-tubulin was from Abcam YL1/2 (ab6160). Rabbit anti-total Pol II CTD and anti-CstF77 have been described (Schroeder et al. 2000 (link); Glover-Cutter et al. 2008 (link)).
7
Integrin Activation Modulation and Visualization
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Antibodies to human β1 integrin used as ligands were 12G10 (ref. 33 (link); Serotec, MCA 2028, 10 μg ml−1), which stabilizes the active integrin conformation, and 4B4 (Beckman Coulter, 6603113, 10 μg ml−1), which stabilizes the inactive integrin conformation. Both 12G10 and 4B4 are the same isotype (IgG1). Other antibodies used were directed against α-tubulin (YL1/2; Abcam, ab6061, 1:1,000 for IF), ACF7 (CU119; provided by R. K. Liem, 1:2,000 for WB), β1 integrin (JB1A; provided by J. A. Wilkins, 1:1,000 for WB), CKAP5 (E-17; Santa Cruz Biotechnology, sc240235, 1:200 for WB), EB1 (Santa Cruz Biotechnology; H-70, sc-15347, 5 μg ml−1 for IF or 1A11, sc-47704, 1:1,000 for WB), talin (C-20; Santa Cruz Biotechnology, sc-7534, 1:2,000 for WB), vinculin (hVIN-1–FITC conjugate; Sigma-Aldrich, F-7053, 1:500 for IF) and FN (39B6; 5 μg ml−1)54 (link). Actin was visualized with phalloidin–Texas Red conjugate (Life Technologies, T7471, 1:500 for IF) or Lifeact–RFP (provided by R. Wedlich-Söldner). Tubulin was visualized with GFP–ensconsin (provided by S. Woolner). Species-specific fluorescent dye-conjugated secondary antibodies were obtained for IF from Jackson Immunoresearch (1:400), and FN, PDL, cytochalasin D and nocodazole were from Sigma-Aldrich.
8
Immunofluorescence Staining Methods
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For immunofluorescence experiments, different fixation methods were exploited depending on the antibodies used.
For immunocytochemistry of MTs, cells were extracted for 1 min. in pre-warmed extraction buffer (0.3% Triton X-100 and 0.1% glutaraldehyde in BRB80 buffer (BRB80 buffer: 80mM Pipes, 1mM EGTA and 4mM MgCl2, pH 6.8) and subsequently fixed in pre-warmed 4% PFA in PBS for 10min. For immunocytochemistry of cytochrome C, cells were fixed in pre-warmed 4% PFA in PBS for 10min. For immunocytochemistry of EB1, cells were fixed in ice-cold methanol for 10min. After fixation, cells were washed with PBS, permeabilized with 0.25% Triton X-100 in PBS, washed again with PBS and subsequently blocked for 1hr with 3% BSA in PBS.
Cells were incubated with primary antibody diluted in 3% BSA in PBS for 1hr at RT, washed with PBS and incubated with secondary antibody diluted in 3% BSA in PBS for 1hr at RT. After washing with PBS, cells were dipped in MQ, air-dried and mounted on microscopy slides using Prolong Diamond (Molecular Probes). The following primary antibodies were used in this study: Cytochrome C (6H2.B4, BD Biosciences), EB1 (5/EB1 BD Biosciences), acetylated tubulin (6-11B-1, Sigma-Aldrich), alpha-tubulin (EP1332Y, Abcam), alpha-tubulin (B-5-1-2, Sigma), tyrosinated tubulin (YL1/2, Abcam) and detyrosinated tubulin (AB3210, Merck).
For immunocytochemistry of MTs, cells were extracted for 1 min. in pre-warmed extraction buffer (0.3% Triton X-100 and 0.1% glutaraldehyde in BRB80 buffer (BRB80 buffer: 80mM Pipes, 1mM EGTA and 4mM MgCl2, pH 6.8) and subsequently fixed in pre-warmed 4% PFA in PBS for 10min. For immunocytochemistry of cytochrome C, cells were fixed in pre-warmed 4% PFA in PBS for 10min. For immunocytochemistry of EB1, cells were fixed in ice-cold methanol for 10min. After fixation, cells were washed with PBS, permeabilized with 0.25% Triton X-100 in PBS, washed again with PBS and subsequently blocked for 1hr with 3% BSA in PBS.
Cells were incubated with primary antibody diluted in 3% BSA in PBS for 1hr at RT, washed with PBS and incubated with secondary antibody diluted in 3% BSA in PBS for 1hr at RT. After washing with PBS, cells were dipped in MQ, air-dried and mounted on microscopy slides using Prolong Diamond (Molecular Probes). The following primary antibodies were used in this study: Cytochrome C (6H2.B4, BD Biosciences), EB1 (5/EB1 BD Biosciences), acetylated tubulin (6-11B-1, Sigma-Aldrich), alpha-tubulin (EP1332Y, Abcam), alpha-tubulin (B-5-1-2, Sigma), tyrosinated tubulin (YL1/2, Abcam) and detyrosinated tubulin (AB3210, Merck).
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