Alexa fluor 594 conjugated goat anti mouse antibody

BeWo cells were fixed with methanol and incubated with anti-E-cadherin antibody (1:200, #3195, CST) and AlexaFluor 594-conjugated goat anti-mouse antibody (Thermo Fisher Scientific) to distinguish cell surfaces. The nuclei were counterstained with 4′,6-diamino-2-phenylindole 2HCl (DAPI). The number of nuclei in syncytiotrophoblasts and total number of nuclei were counted in five randomly selected microscopic areas per sample, and the fusion index was calculated [(number of nuclei in syncytia/total number of nuclei) × 100] in three independent experiments^{29 (link)}.

BE(2)-C, CHP134 and Kelly cells were seeded into Lab-Tak™ Chamber slides (Thermo Scientific, Pittsburgh, PA), and treated with vehicle control, 500nM, 8nM vincristine, or combination for 24 hours. Cells were then fixed for 15 minutes in 4% paraformaldehyde, blocked with 2% horse serum, and incubated with mouse anti-α-tubulin antibody (Sigma, Ct Louis, MO), Alexa Fluor 594-conjugated goat anti-mouse antibody (Thermo Scientific) and DAPI (Vector Laboratories, Burlingame, CA, USA). Cell images were captured under a fluorescence microscope (Zeiss, Oberkochen, Germany), and cells with aberrant mitotic spindles were quantified.

Immunofluorescence to characterize PPAR expression in brain sections was performed using sequential day staining. After 3 × 10 min. washes in PBS, nonspecific binding was blocked by incubating for 1 h at room temperature in a blocking buffer containing dilution media, 5% normal serum, and 0.5% Tx-100. To assess colocalization of PPARs and TH, after washing and blocking, the sections were first incubated overnight at 4 °C with the primary antibodies—anti-PPAR-α (1:100;11540-1-AP, Proteintech), anti-PPAR-β/δ (1:100;1056-2-AP, Proteintech), or anti-PPAR-γ (1:100;16643-1-AP, Proteintech), and anti-TH (AB152, EMD Millipore). For the secondary antibody-antigen reaction, the tissues were rinsed in PBS and then incubated in Alexa Fluor 488-conjugated donkey anti-rabbit antibody (1:300; Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse antibody (1:300; Invitrogen) for 1 h at room temperature. The quantification of PPAR positive cells was performed while using the Olympus BX-51 microscope. PPAR isotype and TH dual-labeled cells were quantified in the SN and VTA.

For microscopy experiments, the myc/mito tagged testin variants and GFP tagged testin variants were co-expressed in HeLa cells. Immunofluorescent staining was performed after fixation of the cells. The myc/mito tagged testin variants were detected using a primary mouse anti-c-myc antibody (9E10, Millipore), and a secondary Alexa Fluor^® 594 conjugated goat anti-mouse antibody (Invitrogen). Cells were imaged using a Zeiss LSM 510 Meta laser scanning confocal microscope (Carl Zeiss, Jena, Germany) with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. Images were processed using ZEN 2009 software. To monitor the subcellular localisation of FL-GFP and FL-Y288-GFP, constructs were overexpressed in HeLa cells, fixed and co-stained with a primary mouse anti-vinculin antibody (SIGMA) and a secondary Goat anti-mouse Alexa 594 antibody. Cells were visualised using an epifluorescence microscope (Leica DMRX, HCX PL APO 100x/1.35NA oil immersion lens).

The specific steps of embedding and sectioning experiments of mouse lung tissues were carried out according to previous studies, as were HE and MASSON experiments. The immunohistochemistry staining of lung tissues immunofluorescence analysis of A549, HBE cells or lung tissues were performed as described previously. The secondary antibodies incubated were horseradish peroxidase-conjugated goat anti-mouse IgG for immunohistochemistry, Alexa Fluor 488-conjugated goat anti-rabbit antibody or Alexa Fluor 594-conjugated goat anti-mouse antibody (Invitrogen) for Immunofluorescence. Besides, nuclei were stained with DAPI (Sigma) for Immunofluorescence.

Immunofluorescence analyses of testicular tissues were performed as previously described12 (link). The following primary antibodies were employed: Rabbit anti-β-catenin, rabbit anti-occludin, and mouse anti-CD68. Alexa Fluor 488-conjugated goat anti-rabbit antibody or Alexa Fluor 594-conjugated goat anti-mouse antibody (Invitrogen) was used as a secondary antibody.

Immunofluorescence analyses of LR-MSCs or lung tissues were performed as described previously [37 (link)]. The following primary antibodies were employed: rat anti-Sca-1, mouse anti-α-smooth muscle actin (α-SMA), rabbit anti-collagen I, mouse anti-CD206, rabbit anti-F4/80, and rabbit anti-Wnt7a. Alexa Fluor 488-conjugated goat anti-mouse antibody, Alexa Fluor 488-conjugated goat anti-rat antibody, Alexa Fluor 594-conjugated goat anti-mouse antibody and Alexa Fluor 594-conjugated goat anti-rabbit antibody (Invitrogen no.A-11001, A-11006, A-11032, and A-11037, respectively, Carlsbad, CA, 1:200 dilution) was used as a secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma no. D9542). The images were observed under confocal fluorescence microscope (Olympus, Tokyo, Japan) with the Z-stack technique (0.8 μm/layer).