Alexa fluor 633 goat anti rat igg h l

Primary antibodies were from Developmental Studies Hybridoma Bank (DSHB): rat anti-Vasa (1:1000), mouse anti-HP1a (1:1000), mouse anti-1B1 (1:1000), and rabbit anti-β-galactosidase (1:1000; Rockland Immunochemicals). Rabbit anti-Piwi (1:1000) and rabbit anti-Ago3 (1:1000) were a kind gift from T. Kai, rabbit anti-Aub (1:1000) and guinea pig anti-Rhino (1:500) were a kind gift from B. Theurkauf. Secondary antibodies were as follows: Alexa Fluor 633 goat anti-rat IgG (H + L) (1:1000), Alexa Fluor 647 goat anti-rat IgM (µchaine) (1:1000), Alexa Fluor 594 goat anti-rat IgM (µchaine) (1:1000), and Alexa Fluor 568 goat anti-rabbit IgG (H + L) (1:1000) from Invitrogen; Alexa Fluor 594 goat anti-rabbit IgG (H + L) (1:1000), Alexa Fluor 594 goat anti-mouse IgG (H + L) (1:1000), and Alexa Fluor 594 goat anti-guinea pig IgG (H + L) (1:1000) from Life Technologies; and GFP-Booster_Atto488 (1:1000) from Chromotek.

NMuMG cells were grown on coverslips, fixed with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 for 15 minutes at room temperature. Subsequently, cells were blocked for 20 minutes at room temperature in blocking solution (10% goat serum, 5% FCS and 0.5% BSA in PBS) followed by incubation with primary antibodies at 4°C overnight. These samples were washed three times with PBS for 10 minutes at room temperature following incubation with fluorochrome-labeled secondary antibody or Phalloidin-633 as well as Hoechst for DNA counterstain (20ng/ml) for 1 hour at room temperature. For mounting of the samples Immu-Mount (Thermo Scientific) reagent was used and samples were imaged with a confocal laser-scanning microscope (SP05, Leica). Data were processed with ImageJ software.
Antibodies used for immunofluorescence were E-Cadherin (13-1900, Invitrogen and 610182, BDTransduction Laboratories), N-Cadherin (610921, BD Transduction Laboratories), ZO-1 (617300, Invitrogen), Alexa Fluor-488 goat anti mouse IgG (H+L) (A11029, Invitrogen); Alexa Fluor-568 goat anti rabbit IgG (H+L) A11011 Invitrogen; and Alexa Fluor-633 goat anti rat IgG (H+L) A21094, Invitrogen, Alexa Fluor 633 Phalloidin (A22284, Invitrogen) was used to stain F-actin.

Following antibodies were used: mouse anti-Cas (610271) (BD Biosciences); rabbit-phospho-Y410Cas (4011S) and phospho-Y249Cas (4014S) (Cell Signaling Technology); mouse anti-vinculin (V9131, Sigma-Aldrich); mouse anti-Crk (610035) (BD Transduction labs); mouse anti-CrkL (05-414) (Upstate); sheep anti-paxillin (AF4259, R and D Systems); rabbit anti-pY31 paxillin (44-720G, Biosource); rat anti-integrin-β1 (9EG7, 553715) and mouse anti-paxillin (610051) (BD Biosciences); rabbit anti-Talin1 (A14168-1-AP), rabbit-anti Kindlin2 (11453-1-AP), and mouse anti-FAK (66258-1-Ig) (Proteintech); rat anti-integrin-β1 (mAB13, MABT821), rabbit anti-integrin β1 (AB1952P), mouse anti-integrin β1 (12G10, MAB2247) (Millipore); rabbit-anti-integrin-β3 (A2542), rabbit-anti-integrin-αv (A2091) (abclonal); rat anti-integrin-β1 (AIIB2), mouse anti-integrin α5β1 (P8D4), and mouse anti-integrin α2β1 (P1E6) (Developmental Studies Hybridoma Bank); AlexaFluor 488 goat anti-rabbit IgG (H+L), AlexaFluor 488 goat anti-mouse IgG (H+L), AlexaFluor 647 goat anti-mouse IgG (H+L), AlexaFluor 647 goat anti-sheep IgG (H+L), and AlexaFluor 633 goat anti-rat IgG (H+L) (Invitrogen); IRDye 680RD goat anti-mouse and IRDye 800CW goat anti-rabbit (LI-COR).