Goat anti mouse igg alexa 555

Cells were fixed in methanol, followed by incubation with a blocking buffer containing 1% BSA and 0.2% Triton X-100 and with a rabbit polyclonal anti-CB2 (1:200, Cayman), a rat anti-F4/80 antibody (1:20, Serotec), a mouse anti-LC3B antibody (1:200, Nano Tools), a guinea pig polyclonal anti-SQSTM1/p62 antibody (1:100, ProGen) or a rabbit anti-HO-1 antibody (1:300) and the appropriate secondary antibodies (goat anti-rabbit IgG Alexa 555 (1:1,000; Invitrogen), goat anti-rat IgG FITC (1:50, Serotec), goat anti-mouse IgG Alexa 555 (1:1,000; Invitrogen), goat anti-Guinea pig IgG Alexa 555 (1:1,000; Invitrogen)) as previously described19 (link). Nuclear staining was performed using Prolong Gold antifade reagent with DAPI (Invitrogen). Fluorescence was imaged on a Zeiss LSM-510 multitracking laser scanning confocal microscope with a Helium/Neon laser at 543 nm and using AxioVision software (Carl Zeiss). No staining was observed when omitting the primary antibody. F4/80- and HO-1-positive cells from 10 fields/conditions were quantified. Results are expressed as percent of HO-1-positive cells per field. The number of SQSTM1/p62 or LC3-positive dots per F4/80-positive cell was quantified from 3–10 fields/condition.

The following primary antibodies were used for immunofluorescence microscopy: human anti-centromere (CREST serum 1:3, Antibodies Inc., 15-234-001; Antibodies Inc., Davis, CA, USA), mouse monoclonal anti-lamin A/C (1:30; Abcam, ab40567; Abcam, Cambridge, UK), monoclonal anti-LAP2α (1:10; clone 15/2, kind gift of Dr. Roland Foisner, Max F. Perutz Laboratories, Vienna, Austria), monoclonal antibody mAb414 (1:2000; Covance, MMS-120R; Covance, Emeryville, CA, USA), monoclonal anti-Hec1 (1:200; clone 3G9, Abcam, ab3613), and rabbit polyclonal anti-LAP2α (1:1000; Abcam, ab5162), polyclonal anti-lamin A (1:500; Sigma-Aldrich, L1293; Sigma-Aldrich, Diegem, Belgium), polyclonal lamin B1 (Abcam, ab16048), polyclonal anti-Sun1 (1:1000, kind gift of Dr. Ulrike Kutay, ETH Zurich, Switzerland), polyclonal anti-Sun2 (1:100; Sigma-Aldrich, HPA001209), polyclonal anti-emerin (1:1000; Bethyl Laboratories, A304-491A; ImTec Diagnostics, Antwerpen, Belgium), as well as polyclonal anti-Nesprin-2 (1:50, kind gift of Dr. Iakowos Karakesisoglou, Durham University, UK). Secondary antibodies were the corresponding goat anti-mouse IgG Alexa 568 (1:1000; Invitrogen), goat anti-mouse IgG Alexa 555 (1:1000; Invitrogen), goat anti-rabbit IgG Alexa 568 (1:1000; Invitrogen), goat anti-mouse IgG Alexa 633 (1:350; Invitrogen), and goat anti-rabbit IgG Alexa 633 (1:350; Invitrogen).