Alexa fluor 488 conjugated goat anti rabbit

Cardiac fibroblasts were seeded on a confocal dish and fixed with 4% formaldehyde. Cells were then immersed in 0.3% Triton X-100 for 15 min and mounted by blocking solution (10% BSA and 0.1% NaN3) for 1 h at room temperature. After incubation with rabbit polyclonal anti-α-SMA (1:1000), anti-MMP9 (1:200) and anti-MRTF-A (1:500) antibodies overnight at 4 °C, cells were washed with 0.01 M PBS and subsequently incubated with Alexa Fluor 488-conjugated goat anti-rabbit or Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (1:1000, Molecular Probes, Eugene, OR, USA). Cells were observed by using a Carl Zeiss LSM710 confocal microscopy (Carl Zeiss, Inc., Jena, Germany). For each group, three independent tests were required and micrographs of 3–5 fields were taken for each well. All micrographs were taken under the same conditions to make sure the fluorescent intense reflecting the content of the protein.

The procedures of tissue dissection used for fixation and staining were described previously [18 (link), 25 (link)]. Confocal images were taken using an Olympus FV1000 laser scanning confocal microscope, appearing as Z-projections in a few optical frames that together covered the bristle cell. The primary antibodies used were mouse anti-acetylated tubulin monoclonal antibodies (1:250; Sigma), and anti-dGM130 (1:500; Abcam), anti-Sec16 antibodies [26 (link)]. Secondary antibodies included Cy3-conjugated goat anti-mouse (1:100; Jackson Immunoresearch) and Alexa Fluor 488-conjugated goat anti-rabbit (1:100; Molecular Probes) antibodies. For actin staining, we used Alexa Fluor 405-conjugated phalloidin (1:250; Molecular Probes).

Immunofluorescence of PASMC was conducted as previously described [19 (link)]. PASMC was seeded in a six-well plate and grown to 50%–60% confluence. After removal of cell culture media, cells were washed twice by PBS and fixed with 4% polyformaldehyde. PASMC was then incubated with 0.5% triton X-100 T for membrane breaking. After blocking, the cell was incubated with primary antibodies against SM α-actin and Ki-67 (1:1000; Cell Signaling Technology) overnight in dark at 4°C. PASMC was then rinsed by PBS and incubated with relative secondary antibodies (Alexa Fluor 594 F(ab’)-conjugated goat anti-mouse and Alexa Fluor 488-conjugated goat anti-rabbit (1:3000; Molecular Probes Inc., Eugene, Oregon, USA)) in dark for 1 h, respectively. The nuclei were stained with DAPI (5 mg/ml; VECTOR Labs, Burlingame, California, USA) for 5 s in the room temperature. Images were captured with immunofluorescent microscopy (Leica MPS 60, Germany).

Ovaries of superovulated 3.5- to 4-week-old Abhd2^+/+ and Abhd2^–/– mice were fixed overnight at 4°C in 4% PFA, and the ovary of a 1-week-old Abhd2^+/+ female was fixed for 4 h after which they went through a sucrose gradient (10−20−30% sucrose/PBS) and were frozen in OCT. To detect localization of ABHD2 in the ovary, 8-μm sections were placed on charged slides and immediately used for staining, according to standard procedures. In brief, for antigen retrieval, the sections were incubated in 1% SDS solution for 5 min, blocked in 5% BSA for 1 h at room temperature, and incubated overnight at 4°C with rabbit polyclonal anti-ABHD2 (1:300) and mouse monoclonal anti-α-actin (1:500, sc-32251, Santa Cruz Biotechnology). The antibody–antigen complexes were visualized by incubation for 1 h at room temperature with 1:2,000 Alexa Fluor 488-conjugated goat anti-rabbit (Molecular Probes A11008/Jackson ImmunoResearch, 111-485-144) and Cy5-conjugated donkey anti-mouse (Jackson ImmunoResearch, 715-175-150) or anti-rabbit (Jackson ImmunoResearch, 711-015-152) antibodies. The sections were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen, P36935) and imaged using confocal laser scanning microscopy (Olympus Fluoview FV1000).

Rabbit and mouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Primary antibodies used in this study include rabbit and mouse anti-FLAG (Sigma-Aldrich Co., St. Louis, MO); mouse anti-myc, chicken anti-GAPDH, and rabbit anti-D2R (Millipore, Billerica, MA); rabbit anti-zDHHC8 (Santa Cruz Biotechnology); mouse anti-GM130 (BD Biosciences, San Jose, CA); mouse anti-GFP (Life Technologies, Carlsbad, CA), and rabbit anti-phospho-p44/42 MAPK (pERK) and rabbit anti-p44/42 MAPK (total ERK) (Cell Signaling, Beverly, MA). HRP-conjugated goat anti-mouse, goat anti-rabbit and donkey anti-chicken secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). Fluorescent secondary antibodies include Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 555-conjugated goat anti-mouse, (Molecular Probes, Eugene, OR), and Cy3-conjugated donkey anti-mouse (Jackson ImmunoResearch).

Promastigotes were washed twice in PBS, fixed in 1% formaldehyde for 30 min, then permeabilized by addition of 0.1% Triton X-100 in PBS for 10 min. Cells were resuspended in 0.1 M glycine in PBS for a further 10 min before being washed in PBS, and then added to poly-L-lysine-coated slides (Sigma-Aldrich, P4707) and allowed to dry. Following 5 min rehydration in TB buffer [0.1% (v/v) Triton X-100 (Sigma-Aldrich, T8787), 0.1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, A9418) in PBS], antibodies to Leishmania glycosomal antibodies (rabbit anti-GAPDH, anti-TIM and anti-GPDH, diluted 1:1000 in TB buffer), were added to slides and incubated for 1 h at room temperature. After washing slides in PBS, the secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit; Molecular Probes, A-11008) was added to slides at 1:500 in TB buffer and incubated for 1 h in the dark. Finally, cells were mounted in VectaShield mounting medium with DAPI (Vector Laboratories, H-1200), and imaged on a DeltaVision Core microscope (Applied Precision Inc., Issaquah (WA), USA) as above.