Alexa fluor 488 conjugated goat anti mouse igg

To characterize human spermatogonia, we examined the expression of a panel of cell-specific proteins including CD49f, Dazl, VASA, STELLA, and the somatic marker VIMENTIN.
Antibodies and Staining. The following primary antibodies were used: mouse monoclonal biotinylated anti-CD49f (BioLegend), mouse monoclonal anti-Dazl (AbD Serotec), goat polyclonal anti-VASA (R&D Systems), rat monoclonal anti-STELLA (R&D Systems), and mouse monoclonal anti-VIMENTIN (Dako). The following secondary antibodies were used: Cy3 conjugated Streptavidin (Dako), Alexa Fluor-488 conjugated goat anti-mouse IgG (Molecular Probes), Cy3 conjugated rabbit anti-goat IgG (Dianova), and Cy3 conjugated goat anti-rat IgG (Dianova). All staining were costained with DAPI.

Hepatic cells seeded on coverslips in 6-well culture plates were treated for 0–30 min with H₂O₂ and fixed within 15 min with ice-cold methanol-acetone 1:1 for 10 min to determine the time-course of maximum activity of PARPs (Fig 1). The coverslips were incubated overnight at 4°C with the primary mouse monoclonal anti-PAR antibody. Two antibodies were indistinctly used for these immunofluorescence studies: cat # 4335-MC-100 (Trevigen, Gaithersburg, MD) and cat # ALX-804-220-R100 (Enzo Life Sciences, San Diego, CA) [28 (link)], both at a dilution of 1:400. Then, the coverslips were incubated with the secondary antibody (Alexa fluor 488-conjugated goat anti-mouse IgG; Molecular Probes, Eugene, OR; cat # A-11001; dilution 1:1000) at room temperature for 2 h and cell nuclei were counterstained with the dye Hoechst 33342 (Sigma) for 2 min. Finally, the coverslips were mounted on slides using Fluoromount G mounting medium (Southern Biotech, Birmingham, AL), and analysed using an Axiophot microscope (Zeiss, Oberkochen, Germany). To determine the level of PARPs activation, the number of PAR-positive nuclei detected at 400X magnification was counted in a total of 200 nuclei for each condition, from three separate experiments.

Immunofluorescence staining was performed according to our published method (Fan et al., 2010 (link)). The primary antibodies against PLD, YFP, BBS3, BBS1, and BBS5 and the secondary antibodies – Alexa-Fluor594-conjugated goat anti-rabbit IgG and Alexa-Fluor488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) – are listed in the key resources table with their suggested dilutions for immunofluorescence staining. Images were captured with an IX83 inverted fluorescent microscope (Olympus) equipped with a back illuminated scientific CMOS camera (Prime 95B, Photometrics) at 100× amplification and processed with CellSens Dimension (version 2.1, Olympus).

The QH1 monoclonal antibody (dilution 1:4, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) labels all quail hemangioblastic cells except mature erythrocytes (Pardanaud et al., 1987 (link)), including immature, ramified, and reactive microglia (Cuadros et al., 1992 (link)). Because the quail retina is avascular, QH1 only labels microglia in retinal explants (Carrasco et al., 2011 (link); Sierra et al., 2014 (link)). The 39.4D5 monoclonal antibody (dilution 1:100, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) recognizes the transcription factor Islet 1 and labels most ganglion cells and other subsets of retinal neurons (Fischer et al., 2002 (link); Halfter et al., 2005 (link)). In addition, the anti-caspase-3 polyclonal antibody (dilution 1:100, R&D Systems, Minneapolis, MN) was used as marker for apoptotic cells in embryo retina (Borsello et al., 2002 (link)). As secondary antibodies we used Alexa Fluor 488-conjugated goat anti-mouse IgG (dilution 1:1,000, Molecular Probes, Eugene, OR) and Cy3-conjugated goat anti-rabbit IgG (dilution 1:1,000, Healthcare Europe, Freiburg, Germany).

To determine cell surface expression of LDLR by FACS, transfected CHO-ldlA7 cells grown during 48 h were incubated with a mouse primary antibody anti-LDLR (1∶100; 5 mg/L; Progen Biotechnik GmbH, Cat. No. 61087) for 1 h, at room temperature, then, washed twice with PBS-1%BSA and incubated with a secondary antibody Alexa Fluor 488-conjugated goat anti-mouse IgG (1∶100; Molecular Probes; Cat. No. A-11001). As negative controls, non transfected CHO-ldlA7 cells or transfected with the wt LDLR were stained with the same primary anti-LDLR antibody (Progen Biotechnik GmbH, Cat. No. 61087), which is negative for CHO-ldlA7 cells and, with mouse IgG2b, kappa monoclonal [MPC-11]-isotype control (abcam; Cat. No. ab18457) in identical conditions. As shown in Figure S1, isotype control antibody has no specificity for CHO-ldlA7 cells transfected or not with LDLR. For each sample, fluorescence of 10,000 events was acquired for data analysis. All measurements have been performed at least in triplicate. LDLR expression efficiency was corrected using the data of mature protein expression quantified by Western blot and data is shown as the percentage of maximum obtained for wt LDLR.

Colonies derived from Dlk⁺ cells were stained with rabbit anti-albumin (Alb) (GeneTex, San Antonio, TX) and mouse anti-cytokeratin 7 (CK7) (DakoCytomation, Fort Collins, CO) antibodies followed by Alexa Fluor 555-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) and Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes) antibodies, respectively. The absolute number of Alb⁺CK7⁺ bipotent cells in each large colony at day 5 of culture was determined in at least 10 large colonies containing more than 100 cells. To examine cellular apoptosis, cells were stained with an anti-caspase 3 (CASP3; Millipore, Billerica, MA) antibody, followed by incubation with Alexa 555-conjugated IgG (Molecular Probes).