Alexa fluor 594 goat anti mouse igg antibody

The amputated fins were fixed overnight in 4% paraformaldehyde in 0.1 M phosphate-buffered saline at 4 °C, embedded in Tissue-Tek O.C.T compound (Sakura Finetek), and cryosectioned to 14 μm thickness by using a Leica CM3050S^{17 (link)}. The following primary antibodies were used: anti-PCNA mouse monoclonal antibody at 1:1000 (Sigma, #P8825) and phospho-S6 ribosomal protein (Ser240/244) rabbit polyclonal antibody at 1:300 (Cell Signaling, #2215). The following secondary antibodies were used: Alexa Fluor^® 488 goat anti-rabbit IgG antibody at 1:500 (Invitrogen, Life Technologies Corp.) and Alexa Fluor^® 594 goat anti-mouse IgG antibody at 1:500 (Invitrogen, Life Technologies Corp.). 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclei staining at a concentration of 1:1000. To detection apoptotic cells, we performed TUNEL staining using an In situ cell death detection kit (Roche, #11684795910) according to the manufacturer’s instructions. The images were captured using an Olympus FV1000-D confocal microscope with the same exposure times using the FluoView software. Eight optical sections per one cryosection along the z-axis were taken in 0.67 μm intervals, and the captured images were analyzed using ImageJ (NIH).

Poly(ε-caprolactone) (M_w 80 kDa), glutaraldehyde, sorbitan monooleate (Span 80), hexamethyldisilazane (HMDS), sodium dodecyl sulfate (SDS), Dulbecco’s modified eagle’s medium (DMEM/F12) were purchased from Sigma, Singapore. Chloroform (CHCl₃) was purchased from Fisher Scientific Company, Loughborough, UK. Micro BCA Protein Assay Kit was purchased from Thermo Scientific (Rockford, lL, USA). Sodium hydroxide (NaOH) was bought from Merck (Singapore). Nerve growth factor (NGF 7S) and ChemiKine Nerve Growth Factor Sandwich ELISA kit were purchased from Millipore, Singapore. Rat pheochromocytoma (PC12) cells in the adherent type [PC12 Adh (ATCC^® CRL1721.1™)] were obtained from ATCC, Manassas, VA, USA, while fetal bovine serum (FBS), horse serum (HS), and trypsin/EDTA were purchased from Invitrogen/Gibco, Grand Island, NY, USA. [3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was purchased from Promega, Madison, WI, USA. Alexa Fluor 594 Goat anti-Mouse IgG antibody was obtained from Invitrogen, Eugene, OR, USA.

The cells were fixed using 3.7% paraformaldehyde for 10 minutes at room temperature. WT and DN Rabs were detected using 556 nm excitation wavelengths in orange color [77 (link)]. Caspase-1 expression was analyzed using primary mouse monoclonal anti-caspase-1 antibody (sc-392736; Santa Cruz Biotechnology, 1:300) and secondary Alexa Fluor^® 594 Goat Anti-Mouse IgG antibody (A-11005, Invitrogen, 1:1000). To demonstrate proteins localizations, 488 nm excitation wavelength in green color was used for Rabs [78 (link),79 (link)], while 590 nm excitation wavelengths in red color were used for Caspase-1. The nucleus was visualized using DAPI (D1306, Termofisher, 300 nM). Images were acquired using confocal laser scanning microscopy on an LSM 700 (Zeiss); ZEN 3.0 Black was used for image processing (Zeiss). The percentages of fusion protein-expressing cells were quantified using NIH ImageJ software version 1.52a.

To optically detect dopaminergic neurons of mouse brain, we performed immunostaining of TH in the SNpc. Brain slices were blocked with 1% normal goat serum for 30 min and incubated with a mouse anti-TH monoclonal antibody (Santa Cruz Biotechnology, cat# sc-25,269, dilution 1:100 in 0.2% PBST) for 18 hr at 4 °C. After being washed in 0.2% PBST, the samples were incubated with Alexa Fluor 594 goat anti-mouse IgG antibody (Thermo Fisher Scientific, dilution 1:100 in 0.2% PBST) for 2 hr at RT. After being washed in 0.2% PBST, the samples were mounted using VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). The specimens were observed using a fluorescence microscope (model BZ-X700; Keyence, Osaka, Japan). The relative density of TH-positive signals in the SNpc was measured quantitatively using ImageJ software.

Human induced pluripotent stem cell-derived cardiomyocyte embedded patches were subjected to immunofluorescence staining at day 1, 7, and 14 after cell seeding. Briefly, fixed samples were permeabilized with 0.1% (v/v) Triton X-100 (Sigma-Aldrich) in PBS for 15 min, blocked with 4% normal goat serum (Abcam, Cambridge, UK) for 1 h, incubated with mouse anti-cardiac troponin T monoclonal antibody (1:300, Thermo Fisher Scientific) overnight at 4 °C and then incubated with Alexa Fluor 647 goat anti-mouse IgG antibody (1:200, Thermo Fisher Scientific) for visualization. The patches were also incubated with mouse anti-⍺-actinin antibody (1:100, Millipore-Sigma, Burlington, MA, USA) overnight at 4 °C and then incubated with Alexa Fluor 594 goat anti-mouse IgG antibody (1:200, Thermo Fisher Scientific) for visualization. Nuclei were counterstained with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) and examined with confocal microscopy (Fluoview 1000, Olympus Corporation, Tokyo, Japan).

4 µm sections were mounted on glass slides pre-coated with silane, deparaffined and rehydrated. Antigen retrieval was achieved by Tris-EDTA buffer pH 9 using microwave for 15 min at 600 W then sections were incubated with 10% (v/v) normal horse serum for blocking. Sections were incubated with LYVE-1 antibody diluted at 1∶2000 with PBS and 3β-HSD antibody (ab75710; Abcam, Cambridge, UK) as a steroidogenic cell marker diluted 1∶1000 with PBS for 1 h at RT. Negative control sections were incubated with normal rabbit serum and normal mouse serum diluted by PBS. Subsequently, the sections were incubated with Alexa Fluor 488 goat anti-rabbit IgG antibody (A-11008; Life Technologies, Carlsbad, CA, USA) and Alexa Fluor 594 goat anti-mouse IgG antibody (A-11005 Life Technologies, Carlsbad, CA, USA) for 1 h at RT. Nuclei were visualized using ProLong Gold including DAPI (P36935; Life Technologies, Carlsbad, CA, USA). Fluorescent images were captured using FSX100 and merged using cellSens (Olympus, Tokyo, Japan).