Primary antibodies used were: Wdr1 (G-13, Santa Cruz), 1:100; Destrin (ab11072, abcam), 1:100; cofilin (ab42824, abcam), 1:5,000; HPRT (ab10479, abcam), 1:5,000; GAPDH (ab8245, abcam), 1:5,000; GFP (ab13970), 1:2,000; myosin II (PRB-440P, Covance), 1:200; pericentrin (PRB-432C, Covance), 1:1,000; E-cadherin (ECCD-1, M. Takeichi, RIKEN, Japan), 1:500; CD104 (β4 integrin, 553745 BD-Pharmingen), 1:500; P-cadherin (P-cad, Zymed), 1:1,000; Keratin14 (PRB-155P, Covance), 1:1,000; keratin 10 (PRB-159P, Covance), 1:1,000; Celsr1 (Fuchs lab), 1:250; keratin 6 (Rb 415, Millipore), 1:500; PAR3 (07-330, Millipore), 1:100; phospho-S3-Cofilin (77G2, Cell Signaling), 1:5,000; Rab11 (D4F5, Cell Signaling), 1:500; β-actin (AC-15, Sigma), 1:5,000; γ-actin (2-2.1.14.17, Sigma), 1:5,000; α-tubulin (DM1A, Sigma), 1:1,000; acetylated tubulin (6-11B-1, Sigma), 1:1,000. Secondary antibodies conjugated to FITC, rhodamine (Jackson Immunoresearch), or Alexa Fluor 488/546/647 (Invitrogen) were diluted 1:1,000 (sagittal sections) or 1:200 (whole mount), and samples were incubated 1 h–overnight. F-actin was labelled with phalloidin–Alexa Fluor 546/647 (Invitrogen).
Alexa fluor 488 546 647
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488/546/647 are fluorescent dyes commonly used in biological research applications. They are available in three different wavelengths (488nm, 546nm, and 647nm) and can be used for various labeling and detection techniques, including flow cytometry, microscopy, and immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
F actin, by Thermo Fisher Scientific (1 mentions)
α tubulin dm1a, by Merck Group (1 mentions)
G actin, by Merck Group (1 mentions)
Destrin, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Keratin 14 prb 155p, by Fortrea (1 mentions)
Prb 440p, by Fortrea (1 mentions)
Rabbit anti apkcζ c20, by Santa Cruz Biotechnology (1 mentions)
Fitc conjugated anti gfp, by Abcam (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Hb 243, by American Type Culture Collection (1 mentions)
Snaka51, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
3 protocols using alexa fluor 488 546 647
1
Multi-Antibody Immunofluorescence Imaging
2
Immunofluorescent Analysis of Ovarian Proteins
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Ovaries were dissected in PBS, fixed for 20 min in 4% paraformaldehyde (PFA) in PBS, washed for 45 min in PBS/0.1% Triton X‐100 (PBST) and blocked for 30 min in 5% normal goat serum (NGS)/PBST. Primary antibodies were diluted in NGS/PBST, and samples were incubated overnight at 4°C.
Primary antibodies used were FITC‐conjugated anti‐GFP (1:400; Abcam), rat anti‐Crb (1:300, a gift from U.Tepass), anti‐Ex (gift from R. Fehon), rabbit anti‐Kib (1:200, Genevet et al, 2010 (link)), rabbit anti‐aPKCζ (C20) (1:250 Santa Cruz), anti‐pWts (gift from K. Irvine), anti‐Rab5 (DSHB), anti‐Rab7 (DSHB), anti‐Rab11 (DSHB), anti‐alpha‐spec (DSHB). Secondary antibodies (goat Alexa Fluor 488, 546, 647; Invitrogen) were used at 1:500 for 2 h at RT, and samples were mounted in Vectashield (Vector Laboratories).
Primary antibodies used were FITC‐conjugated anti‐GFP (1:400; Abcam), rat anti‐Crb (1:300, a gift from U.Tepass), anti‐Ex (gift from R. Fehon), rabbit anti‐Kib (1:200, Genevet et al, 2010 (link)), rabbit anti‐aPKCζ (C20) (1:250 Santa Cruz), anti‐pWts (gift from K. Irvine), anti‐Rab5 (DSHB), anti‐Rab7 (DSHB), anti‐Rab11 (DSHB), anti‐alpha‐spec (DSHB). Secondary antibodies (goat Alexa Fluor 488, 546, 647; Invitrogen) were used at 1:500 for 2 h at RT, and samples were mounted in Vectashield (Vector Laboratories).
3
Integrin-targeting Antibodies for Microscopy
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Function blocking antibodies directed against α5 integrin, clone IIA1 was from BD Biosciences and against αv integrin, clone 69.6.5 was produced and purified as previously described [34] . Following antibodies were used for confocal microscopy. Conformation dependent LIBS anti-α5 antibody clone SNAKA-51 was from Millipore. Phycoerythrin-conjugated anti-α5 antibody (clone SAM-1) was from BioLegend. Anti-β1 antibody clone TS2/16 was produced from the hybridoma cell line TS2/16.2.1 (HB-243, ATCC) and purified on protein-G-sepharose column (Pierce). Anti-αv antibody, clone PLW7, was from Santa Cruz Biotechnology, anti-fibronectin antibody, clone 10, was from BD Biosciences, anti-pFAK-tyr397, clone 141-9 from Life Technologies. Fluorescently labeled secondary antibodies were purchased from Invitrogen (Alexa Fluor -488; -546; -647). Dapi was purchased from Santa Cruz Biotechnology. Human fibronectin stabilized solution was purified as described in Reference 35. Cell culture medium and reagents were from Lonza. All other reagents were of molecular biology quality.
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